Compositions and methods for treating diseases by inhibiting exosome release

ABSTRACT

A method for treating a cancer comprises administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising an anti-cancer agent having at least one secretion modifying region (SMR) peptide from HIV-1 Nef fused to at least one cell-penetrating peptide (CPP) or at least one Clusterin (Clu)-binding peptide (Clu-BP).

This application is a Continuation of U.S. application Ser. No.16/226,283, filed on Dec. 19, 2018, which is a Continuation-in-Part ofU.S. application Ser. No. 16/030,430, filed on Jul. 9, 2018, now U.S.Pat. No. 10,544,193, which is a continuation of U.S. application Ser.No. 15/383,454, filed Dec. 19, 2016, now U.S. Pat. No. 10,040,831. Theentirety of the aforementioned applications is incorporated herein byreference.

This application was made with government support under certain grantsawarded by NIH. The government has certain rights in the application.

FIELD

The present disclosure generally relates to compositions and methods formedical treatment, and in particular, to methods for treating cancersand infectious diseases by inhibiting exosome release.

BACKGROUND

Membrane exosomes are spherical membrane microvesicles, generally lessthan 200 nm in diameter. The exosomes are composed of a lipid bilayercontaining a cytosolic fraction. Particular membrane vesicles are morespecifically produced by cells, from intracellular compartments throughfusion with the cytoplasmic membrane of a cell, resulting in theirrelease into the extracellular biological fluids of an organism or intocell culture media. These exosomes may be released in a number of ways.The classical secretory pathway processes mainly traditional membranesignals bearing receptors through the endoplasmic Reticulum (ER)membrane (Lee et al., (2004) Annu. Rev. Cell Dev. Biol. 20, 87-123).

Secretory proteins are packaged into transport vesicles, delivered tothe Golgi apparatus, and eventually released of into the extracellularspace. Alternatively, nonclassical secretory pathways mediatetranslocation of cytosolic, nonsignal bearing molecules into theextracellular space (Lippincott-Schwartz et al., (1989) Cell 56,801-813; and Misumi et al., (1986) J. Biol. Chem. 261, 11398-11403). Twoof these involve intracellular vesicles of the endocytic membranesystem, such as secretory lysosomes (Muesch et al., (1990) TrendsBiochem. Sci. 15, 86-88) and exosomes (Johnstone et al., (1987) J. Biol.Chem. 262, 9412-9420), the latter ones being internal vesicles of lateendosomes or multivesicular bodies (MVB). Lysosomal contents gain accessto the exterior of cells when specialized endocytic structures such assecretory lysosomes of cytotoxic T lymphocytes, fuse with the plasmamembrane. Lumenal contents of late endocytic structures are releasedinto the extracellular space when MVBs fuse with the plasma membraneresulting in release of the internal multivesicular endosomes into theextracellular space (called exosomes) along with their cargo molecules.Other nonclassical pathways involve direct translocation of cytosolicfactors across the plasma membrane using protein conducting channels ora process called membrane blebbing (Nickel, W. (2005) Traffic. 6,607-614). Membrane blebbing is characterized by shedding of plasmamembrane-derived microvesicles into the extracellular space.

Exosome release has been demonstrated from different cell types invaried physiological contexts. It has been demonstrated that tumor cellssecrete exosomes, such as exosomes in a regulated manner, which cancarry tumor antigens that can be presented to antigen presenting cells(Patent Application No. WO99/03499). In addition, FasL or TNF containingexosomes are known to cause a state of immune privilege/immunesuppression which can promote tumor growth. Similarly, virus-infectedcells, including those infected by HIV are known to releaseNef-containing exosomes (Guy et al., (1990) Virology 176, 413-425; andCampbell et al., (2008) Ethn. Dis. 18, S2-S9), which serve to suppressthe immune system allowing HIV to survive. Exosome secretion has beenshown to utilize the same endosomal trafficking pathway involved invirion release from infected cells (Sanfridson et al., (1997) Proc.Natl. Acad. Sci. U.S.A 94, 873-878; and Esser et al., (2001) J Virol.75, 6173-6182). Tumors are known to release large numbers of exosomes,which can cause immune suppression through immune cell killing ordysregulation, thereby promoting a state of immunosuppression thatallows for rapid tumor growth (Lindner K. et al., 2015,Salido-Guadarrama I. et al., 2014). Similarly, HIV infections result inhigh numbers of exosomes, which appears to contribute to a state ofimmune privilege/suppression which ultimately could lead to AcquiredImmune Deficiency Syndrome (AIDS). The exosome secretion pathway isinvolved in the regulation of cancer homeostasis, the immune system andvirion release of infected cells. In view of the foregoing, there is aneed in the art for compositions and effective methods of treatment forinhibiting exosome release.

SUMMARY

One aspect of the present disclosure relates to a multipartite peptidethat inhibits the release of exosomes from cells. The peptide containsat least one secretion modifying region (SMR) peptide from HIV-1 Nef andat least one Clusterin (Clu)-binding peptide (Clu-BP) and/or cellpenetrating peptide (CPP).

In one embodiment, a method for treating a cancer, comprisesadministering to a subject in need of such treatment an effective amountof a pharmaceutical composition comprising a multipartite peptidecomprising at least one secretion modifying region (SMR) peptide fromHIV-1 Nef fused to at least one cell-penetrating peptide (CPP) or atleast one Clusterin (Clu)-binding peptide (Clu-BP).

In one embodiment, the SMR peptide is fused to at least one CPP. Inanother embodiment, and SMR peptide is fused to 2, 3, 4 or 5 CPPsequences. In a particular embodiment, the peptide has an SMR peptidemotif at its N-terminal end and a CPP peptide motif at its C-terminalend. In another embodiment, the peptide has a CPP peptide at itsN-terminal end and an SMR peptide at its C-terminal end.

In another embodiment, the SMR peptide is fused to at least one Clu-BP.In another embodiment, the SMR peptide comprises 2, 3, 4 or 5 Clu-BPsequences. In a particular embodiment, the peptide has an SMR peptidemotif at its N-terminal end and a Clu-BP peptide motif at its C-terminalend. In another embodiment, the peptide has a Clu-BP peptide at itsN-terminal end and an SMR peptide at its C-terminal end.

In another embodiment, the SMR peptide comprises at least one CPP and atleast one Clu-BP.

In one embodiment, the SMR peptide comprises an amino acid sequenceselected from the group consisting of VGFPV (SEQ ID NO: 1), VGFPVAAVGFPV(SEQ ID NO: 2), and NXNVGFPVAAVGFPV (SEQ ID NO: 36).

In another embodiment, the at least one CPP comprises the amino acidsequence GRKKRRQRRRPPQ (SEQ ID NO: 38).

In some embodiments, the at least one Clu-BP comprises an amino acidsequence selected from the group consisting of HPLSKHPYWSQP (SEQ ID NO:3), NTYWSQLLHFQT (SEQ ID NO: 4) and SHALPLTWSTAA (SEQ ID NO: 5).

In one embodiment, the subject has breast cancer.

In another embodiment, the subject has leukemia.

In another embodiment, the method further comprises the step ofadministering to the subject a second anti-cancer agent.

In a particular embodiment, the second anti-cancer agent is paclitaxelor cisplatin.

In another embodiment, the second anti-cancer agent is a mortalininhibitor.

In a particular embodiment, the mortalin inhibitor is MKT-077,Omeprazole or 5-(N,N-dimethyl)amiloride (DMA).

In another aspect, a polynucleotide encodes the multipartite peptidedescribed herein.

In one embodiment, the polynucleotide is an expression vector operablylinked to a regulatory sequence.

In another embodiment, a cell comprises the expression vector.

In another aspect, a pharmaceutical composition comprises an anti-canceragent comprising at least one secretion modifying region (SMR) peptidefrom HIV-1 Nef fused to at least one cell-penetrating peptide (CPP) orat least one Clusterin (Clu)-binding peptide (Clu-BP).

In one embodiment, the pharmaceutical composition further comprises asecond anti-cancer agent.

In a more particular embodiment, the second anti-cancer agent isselected from the group consisting of mortalin (Hsp70) inhibitors,alkylating agents, anthracycline antibiotics, anti-metabolites,detoxifying agents, interferons, polyclonal or monoclonal antibodies,EGFR inhibitors, HER2 inhibitors, histone deacetylase inhibitors,hormones or anti-hormonal agents, mitotic inhibitors,phosphatidylinositol-3-kinase (PI3K) inhibitors, Akt inhibitors,mammalian target of rapamycin (mTOR) inhibitors, proteasomal inhibitors,poly(ADP-ribose) polymerase (PARP) inhibitors, Ras/MAPK pathwayinhibitors, centrosome declustering agents, multi-kinase inhibitors,serine/threonine kinase inhibitors, tyrosine kinase inhibitors,VEGF/VEGFR inhibitors, taxanes or taxane derivatives, aromataseinhibitors, anthracyclines, microtubule targeting drugs, topoisomerasepoison drugs, and combinations thereof.

In another embodiment, the multipartite peptide is pegylated. In anotherembodiment, the multipartite peptide is incorporated into, onto, orotherwise associated with a nanoparticle. In certain particularembodiments, the nanoparticle is linked to a CPP peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the application will beapparent upon consideration of the following detailed description, takenin conjunction with the accompanying figures and paragraphs. Thefollowing are brief descriptions of the drawings herein, whichillustrate certain aspects and embodiments of the present application,but are not considered limiting in any way.

FIG. 1 shows that SMRwt peptide antagonists inhibit proliferation ofMCF-7 and MDA-MB-231 breast cancer cells but not non-tumorigenic cells.Cells were incubated with peptides at varying dosage (0-1120 nM) for 24hr, after which proliferation was measured by MTT assay. Results ofthree independent experiments are shown. Panel A: Proliferation of MCF-7breast cancer cells. Panel B: Proliferation of MDA-MB-231 breast cancercells. Panel C: Proliferation of non-tumorigenic MCF-10A cells. Lighterdots indicate PEG-SMRwt peptide, darker dots indicate PEG-SMRwt-CLUpeptide, and triangles indicate PEG-SMRmut peptide.

FIG. 2 shows that PEG-SMRwt-CLU peptide antagonist and chemotherapeuticsinduced cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cells.Cells were treated 48 hr with SMR peptides, alone or in combination withpaclitaxel or cisplatin, and were assayed by Cellometer imagingcytometry, indicating percentage of MCF-7 (Panel A) and MDA-MB-231(Panel B) in various cell cycle phases. Results of two independentexperiments are shown. Significant differences relative to untreatedcontrol are indicated as follows: *p<0.01, **p<0.001 for MCF-7 cells,and *p<0.02, **p<0.01, ***p<0.0001 for MDA-MB-231 cells.

FIG. 3 shows that PEG-SMRwt-CLU peptide antagonist increasedcytotoxicity in MCF-7 but not in MDA-MB-231 breast cancer cells.Percentage of apoptotic cells (Panel A) MCF-7 and (Panel B) MDA-MB-231,as determined by Annexin V-FITC assay of cells treated for 48 hr withpeptide alone or combined with paclitaxel or cisplatin. Error barsrepresent mean±SD of four independent experiments. Significantdifferences relative to SMRwt peptide are indicated as follows: *p<0.01,***p<0.0001.

FIG. 4 shows that PEG-SMRwt-CLU peptide antagonist blocks exosomerelease from MCF-7 and MDA-MB-231 cells. Cells were treated for 48 hrwith peptide alone or combined with paclitaxel or cisplatin. Panels Aand B show relative level of exosomes released from MCF-7 and MDA-MB-231cells respectively, determined by AchE assay. Error bars represent meanSD of four independent experiments. Significant differences relative toSMRwt peptide: *p<0.01, **p<0.001, ***p<0.0001 for MCF-7 cells; and*p<0.01 for MDA-MB-231 cells. Panels C and D show relative numbers ofexosomes released by MCF-7 and MDA-MB-231 cells respectively, asdetermined by Nanosight measurement. Error bars represent mean SD of twoindependent experiments. Significant differences relative to SMRwtpeptide: *p<0.01, **p<0.001, ***p<0.0001 for MCF-7 cells and *p<0.03,**p<0.02, ***p<0.01 and ****p<0.001 on MDA-MB-231 cells.

FIG. 5 shows that exosome-specific proteins can be detected on exosomesfrom MCF-7 breast cancer cells. Cells were treated for 48 hr with SMRwtpeptide alone or combined with paclitaxel or cisplatin. Panel A:Expression of exosome proteins by Western blot analysis. Panel B:Exosome numbers were measured by NanoSight. Panel C: Densitometryanalysis showing relative intensity of bands. Data represent the mean SDof three independent experiments. Significant differences relative totreatment with peptide are indicated as follows: *p<0.01, **p<0.001,***p<0.0001.

FIG. 6 shows that exosome-specific proteins can be detected on exosomesfrom MDA-MB-231 breast cancer cells. Cells were treated for 48 hr withSMRwt peptide alone or combined with paclitaxel or cisplatin. Panel Ashows Expression of exosome proteins by Western blot analysis. Exosomenumbers were measured by NanoSight (Panel B) and densitometry analysisshows relative intensity of bands (Panel C). Data represent the mean SDof three independent experiments. Significant differences relative totreatment with peptide are indicated as follows: *p<0.01, **p<0.001,****p<0.0001.

FIG. 7 shows that antibody to mortalin inhibits exosome secretion fromMCF-7 breast cancer cells. MCF-7 cells were either transfected withantibodies to mortalin or alpha-tubulin, or treated with SMRwt or SMRmutpeptides. Panel A: Relative exosome release level after 48 hr by AchEassay. Panel B: Relative numbers of exosomes released after 48 hr byNanoSight analysis. Error bars represent the mean SD of threeindependent experiments. Significant differences relative to untreatedcells: *p<0.0001, **p<0.0001.

FIG. 8 shows exosome secretion is decreased in MCF-7 breast cancer cellsby knockdown of mortalin expression. MCF-7 cells were transfected withclones expressing siRNA against either mortalin (HSPA9) or a negativecontrol RNA. Exosomes were isolated and analyzed after 24, 48, 72, and96 hr for changes in level of exosome secretion by AchE assay (Panel A).Significant differences relative to controls are indicated: ***p<0.0001,and exosome secretion by N—Rh-PE (Panel B). Significant differencesrelative to controls are indicated: ***p<0.0001. Panel C: percentage oflive cells remaining at each time point, *p<0.05, **p<0.002,***p<0.0001. Panel D: mortalin and CD63 protein expression levels byWestern blotting. Panel E: Densitometry analysis of Western blot data.Significant differences relative to controls are indicated: *p<0.01,**p<0.001, ***p<0.0001.

FIG. 9, panel A shows synthetic SMRwt-CPPtat (SEQ ID NO: 39) andSMRmut-CPPtat (SEQ ID NO: 41) peptides used for the experiments depictedin FIGS. 8-14. Panel B shows synthetic SMRwt-Clu BP (SEQ ID NO: 37) andSMRmut-Clu BP (SEQ ID NO: 43) peptides used for the experiments depictedin FIGS. 8-14. The SMRwt peptide is highly conserved across all HIV-1clades, HIV-2, and SIV. In Panel B, the peptides further include apolyethylene glycol moiety at the N-terminus, upstream of an asparagineendopeptidase cleavage site.

FIG. 10 shows that SMRwt-CPP peptides reduce proliferation of MCF-7 andMDA-MB-231 breast cancer cells and K562 leukemia cells, but notnon-tumorigenic MCF-10A cells. Cells were incubated with peptides atvarying dosages (0-1120 nM/mL) for 24 hour at 37° C., after whichproliferation was measured by an MTT assay. The results of threeindependent experiments are shown in Panels A-D. Panel A shows reducedproliferation of MCF-7 breast cancer cells with an IC50 of 180 nM/mL;Panel B shows reduced proliferation of MDA-MB-231 breast cancer cellswith an IC50 of 476 nM/mL; Panel C shows no effect on proliferation ofnon-tumorigenic MCF-10A cells as control cells; Panel D shows reducedproliferation of K562 leukemia cells with an IC50 of 907 nM/mL. Thereduced proliferation in Panels A, B and D was observed only in thepresence of wile-type SMR, but not in the presence of the mutant SMR.

FIG. 11 shows that treatment of MCF-7 breast cancer cells, MDA-MB-231breast cancer cells and K562 leukemia cells with PEG-SMRwt-Clu peptide,SMRwt-CPP peptide, or the mortalin inhibitors, MKT-077, Omeprazole or5-(N,N-dimethyl)amiloride (DMA) antagonist blocked exosome release. Eachof these three cell types were treated with PEG-SMRwt-Clu peptide,PEG-SMRmut-Clu peptide, SMRwt-CPP peptide, SMRmut-CPP peptide, MKT-077,Omeprazole or DMA for five days at 37° C. Panels A, B and C show levelsof exosome release from MCF-7 cells, MDA-MB-231 cells, and K562 cells,respectively, as determined by an acetylcholinesterase (AchE) assay.

FIG. 12 shows detection of mortalin, vimentin and the exosome-specificALIX protein in exosomes released from MCF-7 breast cancer cells,MDA-MB-231 breast cancer cells and K562 leukemia cells treated withPEG-SMRwt-Clu, PEG-SMRmut-Clu, SMRwt-CPP, SMRmut-CPP, MKT-077,Omeprazole or DMA for 48 hours at 37° C. Western blot analysis wascarried out using anti-mortalin (Grp-75), anti-Vimentin and anti-Alixantibodies to evaluate mortalin, vimentin and exosomes expression. PanelA shows gel images of the exosome products, mortalin, vimentin and Alixin MCF-7 and MDA-MB-231 breast cancer cells and K562 leukemia cells;Panel B shows band intensities of the exosome products mortalin,vimentin and Alix in MCF-7 and MDA-MB-231 breast cancer cells and K562leukemia cells. Quantitative results from Western blots were obtained bydensitometry analysis of relative band intensities. The data shownrepresent the mean SD of three independent experiments. Significantdifferences relative to treatment with peptide are indicated as follows:*p<0.01, **p<0.001, ***p<0.0001****p, *****p<0.000001,******p<0.0000001, *******p<0.00000001, ********p<0.000000001.

FIG. 13 shows the effects of the mortalin inhibitors MKT-077, DMA andOmeprazole on paclitaxel-(Pac) or cisplatin (Cis)-induced apoptosis incancer cells. Cells were treated for 48 hr with 925 nM/mL of MKT-077 onMDA-MB-231 cells, 500 nM/mL of MKT-077 on MCF-7 cells, 337.5 nM/mL ofMKT-077 on K562 cells, 300 μM/mL of DMA, 200 μM/mL of Omeprazole, 1.6uM/mL of paclitaxel, 2 mg/mL of cisplatin, or combined treatment witheither paclitaxel or cisplatin in combination with each of the threemortalin inhibitors. Panel A: Relative level of apoptosis fromMDA-MB-231 cells; Panel B: Relative level of apoptosis from MCF-7 cells;and Panel C: Relative level of apoptosis from K562 leukemia cells, asdetermined by TUNEL assay. Error bars represent mean SD of fourindependent experiments. Significant differences relative to inhibitorsare indicated as follows: significant differences relative to DMA versusDMA combined Paclitaxel and Omeprazol versus Omeprazol combinedPaclitaxel and Omeprazol combined Cisplatin, respectively, are indicatedas follows: **p<0.002, ***p<0.0002 and *****p<3.68E-06 for MDA-MB-231cells; MKT-077 versus MKT-077 combined Paclitaxel, DMA versus DMAcombined Cisplatin, Omeprazole versus Omeprazole combined Cisplatin,respectively, are indicated as follows: ***p<0.0001, ****p<6.18E-05,*******p<7.75E-08 for MCF-7 cells; Omeprazole versus Omeprazole combinedCisplatin are indicated ***p<0.0009 for K562.

FIG. 14 shows that in the presence of NHS, PEG-SMRwt-Clu and SMRwt-CPPpeptides SMRwt peptides block secretion of mortalin and exosomes andinduce complement-mediated cytotoxicity in K562, MCF-7 and MDA-MB-231cultures. Cells were treated with 280 nM PEG-SMRwt-Clu peptide or 560 nMSMRwt-CPP peptide alone for 60 min at 37° C., followed by treatment with1 μg/mL anti-CXCR4 antibody and 50 μL NHS or NIS for additional 60 minat 37° C. PEG-SMRmut-Clu peptide and SMRmut-CPP peptide were used asnegative controls. The percentage of stained (dead) cells isrepresentative of 3 independent experiments. Panels A-C show relativelevels of cells surviving a complement attack as determined by trypanblue exclusion. Panel A shows percent cell survival following treatmentof MCF-7 breast cancer cells (as indicated); Panel B shows percent cellsurvival following treatment of MDA-MB-231 breast cancer cells; andPanel C shows percent cell survival following treatment of K562 leukemiacells. Cytotoxicity was not observed in cells treated with SMR peptidesalone. Error bars represent mean SD of four independent experiments.Significant differences relative to PEG-SMRwt-Clu peptide and SMRwt-CPPpeptide are indicated as follows: *p<0.01, **p<0.001.

FIG. 15 shows that knockdown of mortalin expression inducedcomplement-mediated cytotoxicity in K562, MCF-7 and MDA-MB-231 cellcultures. Cells were transfected with a vector expressingdouble-stranded mortalin siRNAs. Panel A shows detection of mortalin andα-tubulin following SDS-PAGE/Western blot analysis using anti-mortalinand anti-α-tubulin antibodies; Panel B shows the relative bandintensities corresponding to the detected products in Panel A; Panel Cshows the relative numbers of exosomes released by MCF-7, MDA-MB-231 andK562 cells as determined by NanoSight measurement; Panel D shows thatsiRNA-mediated knockdown of mortalin expression (HSPA9) inducedcomplement-mediated cytotoxicity in the presence of normal human serum(NHS), but not in the presence of heat-inactivated serum (HIS), nor inmock-transfected cells or in cells expressing non-mortalin siRNAs (Neg(“Neg” means an siRNA predicted not to target any known vertebrategene)) regardless of the presence or absence of NHS. Significantdifferences relative to siRNA-Neg transfected cells with NHS andmortalin siRNA transfected cells treated with NHS are indicated asfollows: *p<0.01, **p<0.001.

FIG. 16 shows that treatment of MDA-MB-231 and MCF-7 breast cancer cellswith SMRwt peptides decreased their migration, while similar treatmentof non-tumorigenic MCF-10A breast cells had no effect on migration.MDA-MB-231 and MCF-7 cells were treated with 1120 nM SMR peptide inMCF-7 cells, 280 nM SMR peptide in MDA-MB-231 cells or 3 μM LatrunculinA as control for 24 hours, and cell migration assays were performedaccording to manufacturer's instructions (Calbiochem manual) using afluorescence plate reader set at an excitation wavelength of 485 nm andan emission wavelength of 515 nm. Panels A, B and C show relativefluorescence units of migration from: Panel A: MCF-7 breast cancercells; Panel B: MDA-MB-231 breast cancer cells; and Panel C: MCF-10Anon-tumorigenic breast cells. Error bars represent mean SD of fourindependent experiments. Significant differences relative to SMRwtpeptide are indicated as follows: *p<0.01, ***p<0.0001, ****p<0.00001,*****p<0.000001.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includes“one or more” peptides or a “plurality” of such peptides. With respectto the teachings in the present application, any issued patent or patentapplication publication described in this application is expresslyincorporated by reference herein. Further, where the phrases “in someembodiments . . . ” or “in certain embodiments . . . ” are used, thepresent disclosure should be construed as embracing combinations of anyof the features defining the different embodiments described herein,unless the features are not combinable with one another, are mutuallyexclusive, or are expressly disclaimed herein.

Definitions

As used herein, the terms “SMR peptide” and “SMRwt peptide” refer to apeptide less than 100 amino acids in length comprising one or morecopies of the amino acid sequence, VGFPV (SEQ ID NO: 1). The term“SMRmut peptide” refer to a peptide less than 100 amino acids in lengthcomprising one or more copies of a mutated VGFPV (SEQ ID NO: 1) peptidesequence that abolishes the functional ability of an SMRwt peptide.

As used herein, the phrase “anti-cancer agent” refers to a “smallmolecule drug”, protein or antibody that can reduce the rate of cancercell growth or induce or mediate the death (e.g., necrosis or apoptosis)of cancer cells in a subject (e.g., a human). The phrase “small moleculedrug” refers to a molecular entity, often organic or organometallic,that is not a polymer, that has medicinal activity, and that has amolecular weight less than about 2 kDa, less than about 1 kDa, less thanabout 900 Da, less than about 800 Da or less than about 700 Da. The termencompasses most medicinal compounds termed “drugs” other than proteinor nucleic acids, although a small peptide or nucleic acid analog can beconsidered a small molecule drug. Examples include chemotherapeuticanticancer drugs and enzymatic inhibitors. Small molecules drugs can bederived synthetically, semi-synthetically (i.e., from naturallyoccurring precursors), or biologically.

As used herein, the term “nanoparticle” refers to any particle having anaverage diameter of less than 500 nanometers (nm). In some embodiments,nanoparticles have an average diameter of less than 300 nm, less than100 nm, less than 50 nm, less than 25 nm, less than 10 nm or less than 5nm. In some embodiments, each nanoparticle has a diameter of less than300 nm, less than 100 nm, less than 50 nm, less than 25 nm, less than 10nm or less than 5 nm.

As used herein, the terms “treat” and “treatment” refer to theamelioration of one or more symptoms associated with a disease, such ascancer or an infectious disease; prevention or delay of the onset of oneor more symptoms of the disease; and/or lessening of the severity orfrequency of one or more symptoms of the disease.

The term “cancer” refers to any one of a variety of malignant neoplasmscharacterized by the proliferation of cells that have the capability toinvade surrounding tissue and/or metastasize to new colonization sites,and includes leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ celltumor and blastoma. Exemplary cancers for treatment with the methods ofthe instant disclosure include cancers of the brain, bladder, breast,cervix, colon, head and neck, kidney, lung, non-small cell lung,mesothelioma, ovary, prostate, stomach and uterus, leukemia, andmedulloblastoma.

The term “infectious diseases” includes those pathologic conditions thatarise from viruses, bacteria, fungi and/or parasites that invade anddisrupt the normal function of the mammalian body. Pages 3-147 of theMerck Manual 13th Edition describe some of these conditions and they areincorporated herein by reference.

The phrases “to a patient in need thereof”, “to a patient in need oftreatment” or “a subject in need of treatment” includes subjects, suchas mammalian subjects, that would benefit from administration of themultipartite peptide of the present disclosure for treatment of a cellproliferative disorder.

The terms “therapeutically effective amount”, “pharmacologicallyeffective amount”, and “physiologically effective amount” are usedinterchangeably to mean the amount of a multipartite peptide that isneeded to provide a threshold level of active antagonist agents in thebloodstream or in the target tissue. The precise amount will depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of the composition, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one skilled in the art, based upon the informationprovided herein or otherwise available in the relevant literature.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

A “control individual” is an individual afflicted with the same diseaseas the individual being treated, who is about the same age as theindividual being treated (to ensure that the stages of the disease inthe treated individual and the control individual(s) are comparable).The individual (also referred to as “patient” or “subject”) beingtreated may be a fetus, infant, child, adolescent, or adult human with acell proliferative disorder.

A “small molecule” refers to an organic or inorganic molecule that isnot a polymer, that has medicinal activity, and that has a molecularweight less than 1 kDa. The term encompasses most medicinal compoundstermed “drugs” other than protein or nucleic acids, although a smallmolecule peptide or nucleic acid analog can be considered a “smallmolecule”. Small molecules drugs can be derived synthetically,semi-synthetically (i.e., from naturally occurring precursors), orbiologically. As used herein, the phrase “large molecule” refers to apolymeric protein- or nucleic acid-based product having a molecularweight greater than 1 kDa.

Peptides

One aspect of the present disclosure relates to a multipartiteanti-cancer peptide that inhibits the release of exosomes from cells. Inone embodiment, the peptide comprises at least one secretion modifyingregion (SMR) peptide from HIV-1 Nef and at least one Clusterin(Clu)-binding peptide (Clu-BP) or at least one cell penetrating peptide(CPP).

In some embodiments, the multipartite anti-cancer peptide has 1, 2, 3, 4or 5 SMR peptide sequences. In particular embodiments, the SMR peptidecomprises an amino acid sequence selected from the group consisting ofVGFPV (SEQ ID NO: 1), VGFPVAAVGFPV (SEQ ID NO: 2), and NXNVGFPVAAVGFPV(SEQ ID NO: 36).

In one embodiment, the SMR peptide is fused to at least one Clu-BP. Inanother embodiment, the SMR peptide comprises 2, 3, 4 or 5 Clu-BPsequences. In a particular embodiment, the peptide has an SMR peptidemotif at its N-terminal end and a Clu-BP peptide motif at its C-terminalend. In another embodiment, the peptide has a Clu-BP peptide at itsN-terminal end and an SMR peptide at its C-terminal end.

In some embodiments, the at least one Clu-BP comprises an amino acidsequence selected from the group consisting of HPLSKHPYWSQP (SEQ ID NO:3), NTYWSQLLHFQT (SEQ ID NO: 4) and SHALPLTWSTAA (SEQ ID NO: 5) asdescribed in U.S. Patent Publication No. 2012/0121507.

In another embodiment, the SMR peptide is fused to at least one CPP. ACPP domain enhances the uptake of the multipartite peptide intoeukaryotic cells. Exemplary CPP domains for use in the presentapplication include, but are not limited to, HIV TAT₄₉₋₅₇ peptide, HIVTAT₄₈₋₆₀ peptide (SEQ ID NO: 38), low molecular weight protamine (LMWP)peptide; Chariot™, also known as Pep-1 (Morris et al., Nat. Biotechnol.,19:1173-1176, 2001); Antp₄₃₋₅₈ peptide, MPG (HIV Gp41-SV40 NLS), SAP,MPG R9, MAP, K-FGF, Penetratin, Buforin II, Transportan, Ku70, Prion,pVEC, Pep-1-K, Pep-7, HN-1, TP10, and CP26 (See e.g., Joliot et al.,Nature Cell Biol., 6(3):189-196, 2004 and Heitz et al., Br. J.Pharmacol., 157:195-206, 2009).

In one embodiment, an SMR peptide is fused to 2, 3, 4 or 5 CPPsequences. In a particular embodiment, the peptide has an SMR peptidemotif at its N-terminal end and a CPP peptide motif at its C-terminalend. In another embodiment, the peptide has a CPP peptide at itsN-terminal end and an SMR peptide at its C-terminal end.

In a particular embodiment, the at least one CPP comprises the aminoacid sequence GRKKRRQRRRPPQ (SEQ ID NO: 38).

In certain embodiments, the peptide comprises at least two SMR peptides,at least two Clu-BP peptides, and/or at least two CPP peptides. Inaddition, any of the peptides within an SMR peptide (e.g., SMR peptides,Clu-BP peptides, CPP peptides) may be separated by a spacer peptide.

In certain particular embodiments, the multipartite peptide comprises anamino acid sequence selected from the group consisting ofVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 6), VGFPVAAVGFPVAAHPLSKHPYWSQP (SEQID NO: 7), VGFPVAAVGFPVAAHPLSKHPYWSQPAAHPLSKHPYWSQP (SEQ ID NO: 8),NXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 37), andVGFPVAAVGFPVGRKKRRQRRRPPQ (SEQ ID NO: 39).

In certain embodiments, the multipartite peptide of the presentinvention further comprises one or more spacers between one or morefunctional domains within the multipartite peptide. The spacer isdesigned to facilitate the independent folding of each domain relativeto one another, ensure that the individual domains in the peptide do notinterfere with one another or with the SMR peptide and/or increase theflexibility of the protein and facilitate adoption of an extendedconformation. In some embodiments, the spacer comprises 1 to 50 aminoacids, preferably 2 to 10 amino acids.

In some embodiments, the spacer includes one or more a glycine and/orserine residues to force the spacer to adopt a loop conformation,because the absence of a β-carbon permits the polypeptide backbone toaccess dihedral angles that are energetically forbidden for other aminoacids. In addition, spacers comprising glycine and/or serine have a highfreedom degree for linking of two peptides, i.e., they enable the fusedproteins to fold and produce functional proteins. Other residues thatcan enhance stability and folding include the amino acids alanine,proline, lysine, and combinations thereof. In one embodiment, the spaceris an Ala-Ala dipeptide linker. In another embodiment, the spacer hasthe formula [(Gly)_(n)-Ser/Ala]_(m) (SEQ ID NO: 35) where n is from 1 to4, inclusive, and m is from 1 to 4, inclusive.

In some embodiments, the multipartite peptide includes a mitochondrialpenetrating sequence or a mitochondrial targeting signal sequence tofacilitate uptake of the multipartite peptides into the mitochondriawhere mortalin is localized. Exemplary mitochondrial targeting sequencesinclude the presequence peptide described in U.S. Patent Publication2004/0192627, including the nuclear-encoded human cytochroine c oxidase(COX) subunit VIII (MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 9); theamino-terminal leader peptide of the rat ornithine transcarbamylase(OTC) (MLSNLRILLNKAALRKAHTSMVRNFRYGKPVQC (SEQ ID NO: 10)), thepresequence of cytochrome oxidase subunit IV (MLSLRQSIRFFKPATRTL (SEQ IDNO: 11)), and an Antennapedia α-helical domain, such as RQIKIWFQNRRMKWKK(SEQ ID NO: 12); various mitochondrial targeting peptides described inU.S. Patent Publication No. 2014/0196172, including N-terminalmitochondrial targeting peptides, MFSYLPRYPLRAASARALVRATRPSYRSALLRYQ(SEQ ID NO: 13), MAAWMRSLFSPLKKLWIRMH (SEQ ID NO: 14), MKLLWRLILSRKW(SEQ ID NO: 15), MWWRRSRTNSLRYT (SEQ ID NO: 16), and MLFRLRRSVRLRGLLA(SEQ ID NO: 17); and the N-terminal mitochondrial targeting peptideMWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 18) as described in U.S. PatentPublication No. 2016/0237129. Exemplary mitochondrial targeting signalpeptide sequences directing proteins or peptides to the mitochondriainclude RRIVVLHGYGAVKEVLLNHK (SEQ ID NO: 19), amino acids 74-95 of RatCytochrome P450 2E1 (CYP2E1), the cleavable prepiece from the yeastcytochrome c oxidase IV precursor (MLSLRQDIRFFKPATRTLCSSR (SEQ ID NO:20)), the mitochondrial-targeting signal from the PB2 protein ofinfluenza viruses, the import signal contained within heme lyases, andthe leader peptide of the mitochondrial matrix enzyme ornithinetranscarbamylase (OTC) as described in U.S. Patent Publication No.2014/0142121.

In some embodiments, the multipartite peptide includes a cell targetingdomain for targeting the peptide to specific types of cells, includingtumor cells, virally-infected cells and the like. The targeting domainmay comprise a peptide fused to the multipartite peptide or it may benon-peptide-based domain chemically conjugated to or covalently attachedthereto. Exemplary targeting domains include a peptides, smallmolecules, ligands, antibody fragments, and aptamers. In addition, atargeting domain may be a small molecule (e.g., folate, adenosine,purine) or a large molecule (e.g., peptide or antibody) thatspecifically binds to a desired target cell of interest. In someembodiments, the targeting domain is present at the C-terminal end ofthe multipartite peptide. In other embodiments, the targeting domain ispresent at the N-terminal end of the multipartite peptide.

In some embodiments, the multipartitite peptide comprises a blood-brainbarrier (BBB) entry peptide. Inclusion of a BBB entry peptidefacilitates delivery of the SMR peptide into the brain for treatment ofbrain cancers, such as glioblastoma. Exemplary BBB entry peptidesinclude GGGGHLNILSTLWKYRC (SEQ ID NO: 45; U.S. Patent Publ. No.2018/0073021), TFFYGGSRGKRNNFKTEEYC (SEQ ID NO: 46; Wang et al., Scient.Rep. (2018) 8:12827), RRRRRRRR (SEQ ID NO: 47; Kamei et al., Biol.Pharm. Bull. (2018) 41:546-554), LRKLRKRLLR (SEQ ID NO: 48; McCully etal., Curr. Pharm. Design (2018) 24(13):1366-1376), CGHKAKGPRKGKRK (SEQID NO: 49; McCully et al. (2018)), FKESWREARGTRIERG (SEQ ID NO: 50;McCully et al, (2018)), KSVRTWNEIIPSKGCLR (SEQ ID: 51; McCully et al,(2018)), HAIYPRH (SEQ ID NO: 52; McCully et al, (2018)), TGNYKALHPHNG(SEQ ID NO: 53; McCully et al, (2018)), THRPPMWSPVWP (SEQ ID NO: 54;McCully et al, (2018)), and YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO:55; U.S. Patent Publ. No. 2018/0028677). Additional BBB entry peptidesare described in U.S. Pat. Appl. Nos. 2011/0230416, 2012/0141416, and2013/0108548.

In some embodiments, the multipartite peptide may be linked to animmunoglobulin Fc region. The Fc region can enhance stability and invivo half-life and can facilitate recruitment of Fc receptor-bearingnatural killer cells, macrophages, neutrophils, and mast cells, whichcan stimulate phagocytic or cytotoxic cells to destroy microbes orinfected cells by antibody-mediated phagocytosis or antibody-dependentcell-mediated cytotoxicity. When using antibody-derived targeting agentsor Fc regions, these domains are preferably “humanized” usingmethodologies well known to those of skill in the art.

The multipartite peptide encoded in the expression vector may furtherinclude a cleavage recognition sequence for proteolytic or anendopeptidase cleavage sequence. Incorporation of an endopeptidasecleavage recognition sequences can facilitate site specific cleavage bya suitable endopeptidase present in a eukaryotic or mammalian cell, suchas asparagine endopeptidase, Factor Xa, furin, thrombin, cathepsin B,plasmin, and various matrix metalloproteinases (MMPs), such as MMP2,MMP7, MMP9, or MMP14.

In certain embodiments, placement of a suitable endopeptidase cleavagerecognition sequence can serve to liberate an attached PEG moiety and/orliposomal moiety linked to the peptide, or liberate one or more peptidedomains from one another so that one or more these peptide domains canfunction independently of one another in e.g., their targeted site.

Asparagine endopeptidase, also known as legumain, is a lysosomalcysteine protease that cleaves protein substrates on the C-terminal sideof asparagine, such as Asn-Asp or Asn-Xaa-Asn. In certain embodiments,the multipartite anti-cancer peptide comprises a PEG moiety (e.g., 10Kd) chemically conjugated to a SMR peptide via the asparagineendopeptidase cleavage sequence, NXN (PEG-NAN-SMR-CLU). Exemplarypeptides conjugated to PEG via an NXN sequence include NXNVGFPVAAVGFPV(SEQ ID NO: 36) and NXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 37).

Sequences cleavable by MMP2, MMP7, MMP9, or MMP14 include PLGLAG (SEQ IDNO: 56), PLG-C(me)-AG (SEQ ID NO: 57), RPLALWRS (SEQ ID NO: 21),ESPAYYTA (SEQ ID NO: 22), DPRSFL (SEQ ID NO: 23), PPRSFL (SEQ ID NO:24), RLQLKL (SEQ ID NO: 25), and RLQLK(Ac) (SEQ ID NO: 26). Cathepsin Bis a tumor associated protease that can act upon the dipeptide sequencesvaline-citrulline and Phe-Lys. Furin cleaves the recongition sequenceArg-X-X-Arg (SEQ ID NO: 27), more preferably Arg-X-(Lys/Arg)-Arg (SEQ IDNO: 28). Factor Xa cleaves after the arginine residue in its preferredcleavage site Ile-(Glu or Asp)-Gly-Arg (SEQ ID NO: 29) and willsometimes cleave at other basic residues, depending on the conformationof the protein substrate. The most common secondary site, among thosethat have been sequenced, is Gly-Arg. Thrombin preferentially cleavesbetween Arg and Gly residues in e.g., the sequence LVPRGS (SEQ ID NO:30).

The multipartite peptide of the present disclosure may be chemicallymodified using one or more methods including, but not limited to,amidation, acetylation (including N-terminal acetylation),carboxylation, glycosylation, methylation (e.g., substitution ofα-hydrogens with methyl groups), carbonylation, phosphorylation,PEGylation, dimerization, addition of interchain and/or intrachaindisulfide bonds, addition of trans olefin, derivatization by knownprotecting/blocking groups, circularization, substitution with D aminoacids, linkage to an antibody molecules or other cellular ligands, etc.

The multipartite peptides of the present disclosure can be modified tocontain additional nonproteinaceous moieties that are known in the artand are readily available. Preferably, the moieties suitable forderivatization of the protein are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), polyvinyl alcohol (PVA), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol (PPG)homopolymers, propylene oxide/ethylene oxide copolymers,polyoxyethylated polyols (e.g., glycerol; POG), polyvinyl alcohol, andmixtures thereof. Polyethylene glycol propionaldehyde may haveadvantages in manufacturing due to its stability in water.

Additional modifications include, for example, point mutations,insertions, deletion, truncation, and backbone substitutions, such as NHto NCH₃, In addition, the peptide may be modified by the insertion ofone or more D amino acids. Further, proline analogs in which the ringsize of the proline residue is changed from 5 members to 4, 6, or 7members can be employed. Cyclic groups can be saturated or unsaturated,and if unsaturated, can be aromatic or non-aromatic.

The multipartite peptides may include modifications to, includingincorporation of any of the functional domains described herein at theN-terminal end of the peptide, the C-terminal end of the peptide, orboth. Alternatively, or in addition, modifications at the N-terminal endmay include an acetylated (Ac) residue and/or an amidated (NH₂) residueat the C-terminal end. Where the C-terminus is amidated, the carboxylicacid of the amino acid is converted to an amide, i.e., NH₂—CH₂—C(O)—NH₂.

The multipartite peptide may further contain one or more covalentlyattached functional groups, preferably attached to either or both of theN and C termini of the polypeptide. These covalently attached groups caninclude stabilizers, couplers, ligands, enzymatic substrates and/orcombinations thereof. Preferred groups include acyl groups on the Nterminus and cysteamine (cya) coupling groups on the C terminal end. Tothe latter may be conveniently attached other chemical moieties, e.g.,dyes, ligands, small molecule drugs, proteins, enzymes, enzymaticsubstrates, etc. Alternatives to cya are also known to those of skill inthe art. For stabilizing and/or blocking, e.g., cya may be replaced withan alky group such as methyl or ethyl, which are known to beconveniently positioned onto a —COOH group.

N-terminal modifications additionally may include, but are not limitedto, methylation (i.e., —NHCH₃ or —NH(CH₃)²), adding a1-amino-cyclohexane-carboxylic acid moiety (Chex); and adding acarbobenzoyl group, or blocking the amino terminus with any blockinggroup containing a carboxylate functionality defined by RCOO—, where Ris selected from the group consisting of naphthyl, acridinyl, steroidyl,and similar groups.

A derivitizing group, including, but not limited to, asulfhydryl-containing group or moiety may be positioned at theC-terminus of the multipartite peptide, even when it is not coupled toanother chemical moiety. In one embodiment, the C-terminal end may bemodified with a cysteamide group (—NH—CH₂—CH₂—SH), which can allowfurther coupling to drugs. A cysteamide group is compatible with thepeptide synthesis using the Fmoc strategy and leads to a C-terminalprotected peptide. Alternatively, the peptide can include a C-terminalcysteine residue containing a sulfhydryl (—SH) group that can beoptionally utilized for conjugation to other moieties. In anotherembodiment, the C-terminal end includes a 2,4-diamino-butyric acid (DAB)moiety. C-terminal modifications may further include replacing the freeacid with a carboxamide group or forming a cyclic lactam at the carboxyterminus to introduce structural constraints.

Naturally occurring side chains of the 20 genetically encoded aminoacids (or D amino acids) may be replaced with other side chains withsimilar properties, for instance with groups such as alkyl, lower alkyl,cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amidedi(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic.

Such substitutions can include but are not necessarily limited to: (1)non-standard positively charged amino acids, like: ornithine;N-(4-aminobutyl)-glycine having a lysine side chain attached to the“N-terminus” and aminopropyl or aminoethyl groups attached to the aminogroup of glycine; (2) Non-naturally occurring amino acids with no netcharge and sidechains similar to arginine, such as citrulline, with orwithout methylene groups; (3) non-standard non-naturally occurring aminoacids with OH (e.g., serine), such as, homoserine, hydroxyproline,hydroxyvaline, and penicillamin; (4) proline derivatives, such as,D-Pro, including 3,4-dehydroproline, pyroglutamine, proline withfluorine substitutions on the ring, 1,3-thiazolidine-4-carboxylic acid;(5) Histidine derivative, such as beta-(2-thienyl)-alanine; or (6) alkylderivatives, such as 2-aminobutyric acid, norvaline, norleucine,homoleucine, and alpha-aminoisobutyric acid.

In other embodiments, the C-terminal carboxyl group or a C-terminalester may be induced to cyclize by internal displacement of the —OH orthe ester (—OR) of the carboxyl group or ester respectively with theN-terminal amino group to form a cyclic peptide. For example, aftersynthesis and cleavage to give the peptide acid, the free acid isconverted to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

In other embodiments, the multipartite peptide of the present disclosureis cyclized or includes a desamino or descarboxy residue at the peptidetermini so that there are no terminal amino or carboxyl groups. This candecrease susceptibility to proteases and/or to restrict the conformationof the peptide. C-terminal functional groups of the compounds of thepresent disclosure include amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, and carboxy, and the lower esterderivatives thereof, and the pharmaceutically acceptable salts thereof.The multipartite peptide may be cyclized by adding an N and/or Cterminal cysteine and cyclizing the peptide through disulfide linkagesor other side chain interactions.

In one preferred embodiment, the multipartite peptide (or pharmaceuticalcomposition thereof) has the structure A-B-C-D/E, where A is a PEG, suchas a 10 kD PEG; B is a peptide cleavage linker sequence for anendopeptidase, such as asparagine endopeptidase; C is an SMR-containingpeptide sequence, such as VGFPVAAVGFPV (SEQ ID NO: 2); D is a Clu-BPpeptide sequence, such as HPLSKHPYWSQP (SEQ ID NO: 3); and E is a cellpenetrating peptide (CPP) sequence, such as GRKKRRQRRRPPQ (SEQ ID NO:38).

The multipartite peptides of the present disclosure may be administeredas naked peptides with or without PEG moieties, or they may beincorporated into, onto, or otherwise associated with a suitablecarrier, such as a liposome, nanoparticle, hydrogel, polymeric micelle,microcapsule, virus, bacteriophage, or virus-like particle (VLP).

Exemplary nanoparticles include paramagnetic nanoparticles,superparamagnetic nanoparticles, metal nanoparticles, polymericnanoparticles, nanoworms, nanoemulsions, nanogels, fullerene-likematerials, inorganic nanotubes, dendrimers (such as with covalentlyattached metal chelates), nanocapsules, nanospheres, nanofibers,nanohoms, nano-onions, nanorods, nanoropes and quantum dots. Ananoparticle can produce a detectable signal, for example, throughabsorption and/or emission of photons (including radio frequency andvisible photons) and plasmon resonance. Nanoparticles can bebiodegradable or non-biodegradable.

In certain embodiments, the nanoparticle is a metal nanoparticle, ametal oxide nanoparticle, or a semiconductor nanocrystal. The metal ofthe metal nanoparticle or the metal oxide nanoparticle can includetitanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium,molybdenum, tungsten, manganese, technetium, rhenium, iron, ruthenium,osmium, cobalt, rhodium, iridium, nickel, palladium, platinum, copper,silver, gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanideseries or actinide series element (e.g., cerium, praseodymium,neodymium, promethium, samarium, europium, gadolinium, terbium,dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium,protactinium, and uranium), boron, aluminum, gallium, indium, thallium,silicon, germanium, tin, lead, antimony, bismuth, polonium, magnesium,calcium, strontium, and barium. In certain embodiments, the metal can beiron, ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver,gold, cerium or samarium. The metal oxide can be an oxide of any ofthese materials or combination of materials. For example, the metal canbe gold, or the metal oxide can be an iron oxide, a cobalt oxide, a zincoxide, a cerium oxide, or a titanium oxide. Preparation of metal andmetal oxide nanoparticles is described, for example, in U.S. Pat. Nos.5,897,945 and 6,759,199.

In other embodiments, a polymeric nanoparticle is made from a syntheticbiodegradable polymer, a natural biodegradable polymer or a combinationthereof. Synthetic biodegradable polymers can include, polyesters, suchas poly(lactic-co-glycolic acid)(PLGA) and polycaprolactone;polyorthoesters, polyanhydrides, polydioxanones,poly-alkyl-cyano-acrylates (PAC), polyoxalates, polyiminocarbonates,polyurethanes, polyphosphazenes, or a combination thereof. Naturalbiodegradable polymers can include starch, hyaluronic acid, heparin,gelatin, albumin, chitosan, dextran, or a combination thereof.

In certain particular embodiments, the above-described carriers,including nanoparticles, may be linked to the CPP peptides, targetingpeptides, mitochondrial targeting peptides, and/or BBB entry peptidesdescribed herein to facilitate carrier-mediated delivery of the activeagents described herein.

Peptide-Encoding Polynucleotides

Another aspect of the present disclosure relates to a polynucleotideencoding any of the multipartite peptides described herein. In oneembodiment, the polynucleotide is an expression vector. As used herein,the term “expression vector” refers to a non-viral or a viral vectorthat comprises a polynucleotide encoding the multipartite peptide of thepresent disclosure in which the peptide coding sequences are operablylinked to regulatory sequences sufficient for expressing the peptide ina cell. One type of non-viral vector is a “plasmid”, which includes acircular double-stranded DNA loop into which additional DNA segments canbe ligated. In the present specification, “plasmid” and “vector” can beused interchangeably as the plasmid is the most commonly used form ofvector.

The regulatory sequences may be selected on the basis of the host cellsto be used for expression, such that the design of the expression vectorand inclusion of regulatory sequences depends on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, whether the peptide is to be secreted into theextracellular milieu and the like. The expression vectors of theinvention can be introduced into host cells to direct the expression ofthe multipartite peptide of the present disclosure in vitro forproduction purposes or in vivo for therapeutic purposes.

As used herein, the terms “control sequences” or “regulatory sequences”refer to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The term“control/regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Control/regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, in certain embodiments, the expression vectorencodes a presequence or signal peptide that is operably linked to thepeptide coding sequences for expression as a preprotein thatparticipates in the secretion of the polypeptide. In addition, apromoter or enhancer is said to be operably linked to a coding sequenceif it affects the transcription of the sequence and a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto facilitate translation. Generally, “operably linked” means that theDNA sequences being linked are contiguous and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking these sequences may be accomplishedby ligation at convenient restriction sites or by the use of syntheticoligonucleotide adaptors, primers, and/or linkers are used in accordancewith conventional practices in the art.

In certain cases, these vectors may be engineered to target certaindiseases or cell populations by using the targeting characteristicsinherent to the virus vector or engineered into the virus vector.Specific cells may be “targeted” for delivery of polynucleotides, aswell as expression. Thus, the term “targeting”, in this case, may bebased on the use of endogenous or heterologous binding agents in theform of capsids, envelope proteins, antibodies for delivery to specificcells, the use of tissue-specific regulatory elements for restrictingexpression to specific subset(s) of cells, or both.

In certain embodiments, the expression vector is engineered to directexpression of the peptide ubiquitously or preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe polynucleotide). Thus, in certain embodiments, expression of thepeptide is under the control of a tissue specific or ubiquitouspromoter, such as the CMV promoter or a CMV-chicken beta-actin hybrid(CAG) promoter. In other embodiments, a tissue specific ortumor-specific promoter may be used. Exemplary tissue-specificregulatory elements are known in the art and may include liver-specificpromoter (e.g., albumin promoter), lymphoid-specific promoters,epithelial cell-specific promoters, promoters of T cell receptors andimmunoglobulins, neuron-specific promoters (e.g., the neurofilamentpromoter), pancreas-specific promoters (e.g., insulin promoter), andmammary gland-specific promoters (e.g., milk whey promoter).Developmentally-regulated promoters (e.g., the α-fetoprotein promoter)are also encompassed.

In certain embodiments, the multipartite expression construct may belinked to a mitochondrial targeting peptide or leader sequence tofacilitate uptake of the expression construct into mitochondrial cellsas described in U.S. Patent Publication No. 20040192627. Conventionalprotocols may be used to conjugate the expression construct with themitochondrial targeting peptide, e.g., pGeneGrip™ technology(Genlantis/Gene Therapy Systems, Inc., San Diego, Calif.).Alternatively, the multipartite peptide coding sequence may be fused toa mitochondrial targeting sequence to direct translocation of theexpressed peptide into mitochondria as described above.

In other embodiments, the multipartite peptide coding sequence may befused to a mitochondrial penetrating moiety or mitochondrial targetingsignal sequence as described above. Exemplary nucleic acids that act asmitochondrial penetrating moieties (such as those described in U.S. Pat.No. 5,569,754) include e.g., CCGCCAAGAAGCG (SEQ ID NO: 31),GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 32),ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 33) orACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCT G (SEQ ID NO:34).

In some embodiments, the multipartite peptide coding sequence may befused to a signal peptide domain for secretion of the peptide from cellsexpressing the peptide. The signal peptide sequence is removed from themature peptide as the mature peptide is secreted from the cell. Since agiven signal peptide sequence can affect the level of peptideexpression, a peptide-encoded polynucleotide may include any one of avariety of different N-terminal signal peptide sequences known in theart.

In some embodiments, the expression vector is a viral vector. A viralvectors may be derived from an adeno-associated virus (AAV), adenovirus,herpesvirus, vaccinia virus, poliovirus, poxvirus, a retrovirus(including a lentivirus, such as HIV-1 and HIV-2), Sindbis and other RNAviruses, alphavirus, astrovirus, coronavirus, orthomyxovirus,papovavirus, paramyxovirus, parvovirus, picornavirus, togaviruses andthe like. A non-viral vector is simply a “naked” expression vector thatis not packaged with virally derived components (e.g., capsids and/orenvelopes).

Non-viral expression vectors can be utilized for non-viral genetransfer, either by direct injection of naked DNA or by encapsulatingthe multipartite peptide-encoding polynucleotides in liposomes,nanoparticles, hydrogels, microcapsules, or virus-like particles. Suchcompositions can be further linked by chemical conjugation to targetingdomains to facilitate targeted delivery and/or entry of nucleic acidsinto desired cells of interest. In addition, plasmid vectors may beincubated with synthetic gene transfer molecules such as polymericDNA-binding cations like polylysine, protamine, and albumin, and linkedto cell targeting ligands such as asialoorosomucoid, insulin, galactose,lactose or transferrin.

Alternatively, naked DNA may be employed. Uptake efficiency of naked DNAmay be improved by compaction or by using biodegradable latex beads.Such delivery may be improved further by treating the beads to increasehydrophobicity and thereby facilitate disruption of the endosome andrelease of the DNA into the cytoplasm.

Pharmaceutical Compositions

In some embodiments, a pharmaceutical composition comprises amultipartite peptide comprising at least one SMR peptide from HIV-1 Nef,at least one Clusterin (Clu)-binding peptide (Clu-BP) and apharmaceutically acceptable carrier. In addition, the multipartitepeptide may include any of the above described modification.

In another embodiment, a pharmaceutical composition comprises anexpression vector encoding a multipartite peptide comprising at leastone SMR peptide from HIV-1 Nef, at least one Clusterin (Clu)-bindingpeptide (Clu-BP) and a pharmaceutically acceptable carrier, whereby theencoded peptide is designed to include any of the above describedmodifications.

As used herein, the term “pharmaceutically acceptable” refers to amolecular entity or composition that does not produce an adverse,allergic or other untoward reaction when administered to an animal or ahuman, as appropriate. The term “pharmaceutically acceptable carrier”,as used herein, includes any and all solvents, solubilizers, fillers,stabilizers, surfactants, binders, absorbents, bases, buffering agents,excipients, lubricants, controlled release vehicles, diluents,emulsifying agents, humectants, lubricants, gels, dispersion media,coatings, antibacterial or antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such carriers and agents for pharmaceuticallyactive substances is well-known in the art. See e.g., A. H. KibbeHandbook of Pharmaceutical Excipients, 3rd ed. Pharmaceutical Press,London, UK (2000).

Exemplary carriers or excipients include but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, polymers such as polyethylene glycols, water,saline, isotonic aqueous solutions, phosphate buffered saline, dextrose,0.3% aqueous glycine, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition, or glycoproteins forenhanced stability, such as albumin, lipoprotein and globulin.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the therapeutic agents. In certain embodiments, the pharmaceuticallyacceptable carrier comprises serum albumin.

Formulation characteristics that can be modified include, for example,pH and osmolality. For example, it may be desired to achieve aformulation that has a pH and osmolality similar to that of human bloodor tissues to facilitate the formulation's effectiveness whenadministered parenterally.

Buffers are useful in the present invention for, among other purposes,manipulation of the total pH of the pharmaceutical formulation(especially desired for parenteral administration). A variety of buffersknown in the art can be used in the present formulations, such asvarious salts of organic or inorganic acids, bases, or amino acids, andincluding various forms of citrate, phosphate, tartrate, succinate,adipate, maleate, lactate, acetate, bicarbonate, or carbonate ions.Particularly advantageous buffers for use in parenterally administeredforms of the presently disclosed compositions in the present inventioninclude sodium or potassium buffers, including sodium phosphate,potassium phosphate, sodium succinate and sodium citrate.

Sodium chloride can be used to modify the tonicity of the solution at aconcentration of 0-300 mM (optimally 150 mM for a liquid dosage form).Cryoprotectants can be included for a lyophilized dosage form,principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Other suitable bulking agents include glycine, arginine, can be includedas 0-0.05% polysorbate-80 (optimally 0.005-0.01%).

In one embodiment, sodium phosphate is employed in a concentrationapproximating 20 mM to achieve a pH of approximately 7.0. A particularlyeffective sodium phosphate buffering system comprises sodium phosphatemonobasic monohydrate and sodium phosphate dibasic heptahydrate. Whenthis combination of monobasic and dibasic sodium phosphate is used,advantageous concentrations of each are about 0.5 to about 1.5 mg/mlmonobasic and about 2.0 to about 4.0 mg/ml dibasic, with preferredconcentrations of about 0.9 mg/ml monobasic and about 3.4 mg/ml dibasicphosphate. The pH of the formulation changes according to the amount ofbuffer used.

Depending upon the dosage form and intended route of administration itmay alternatively be advantageous to use buffers in differentconcentrations or to use other additives to adjust the pH of thecomposition to encompass other ranges. Useful pH ranges for compositionsof the present invention include a pH of about 2.0 to a pH of about12.0.

In some embodiments, it will also be advantageous to employ surfactantsin the presently disclosed formulations, where those surfactants willnot be disruptive of the drug-delivery system used. Surfactants oranti-adsorbants that prove useful include polyoxyethylenesorbitans,polyoxyethylenesorbitan monolaurate, polysorbate-20, such as Tween-20™,polysorbate-80, polysorbate-20, hydroxycellulose, genapol and BRIJsurfactants. By way of example, when any surfactant is employed in thepresent invention to produce a parenterally administrable composition,it is advantageous to use it in a concentration of about 0.01 to about0.5 mg/ml.

Additional useful additives are readily determined by those of skill inthe art, according to particular needs or intended uses of thecompositions and formulator. One such particularly useful additionalsubstance is sodium chloride, which is useful for adjusting theosmolality of the formulations to achieve the desired resultingosmolality. Particularly preferred osmolalities for parenteraladministration of the disclosed compositions are in the range of about270 to about 330 mOsm/kg. The optimal osmolality for parenterallyadministered compositions, particularly injectables, is approximately300 mOsm/kg and achievable by the use of sodium chloride inconcentrations of about 6.5 to about 7.5 mg/ml with a sodium chlorideconcentration of about 7.0 mg/ml being particularly effective.

Multipartite peptides can be stored as a lyophilized powder underaseptic conditions and combined with a sterile aqueous solution prior toadministration. The aqueous solution used to resuspend the peptides cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physical conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, as discussed above.Alternatively, the multipartite peptides can be stored as a suspension,preferable an aqueous suspension, prior to administration.

In certain preferred embodiments, the multipartite peptide in thepharmaceutical composition is pegylated. PEGylation is a process forcovalently attaching polyethylene glycol polymer chains to anothermolecule, normally a drug or therapeutic peptide/protein. PEGylation canbe achieved by incubation of a reactive derivative of PEG with themultipartite peptide. The covalent attachment of PEG to a multipartitepeptide can “mask” the multipartite peptide from the host's immunesystem (reduced immunogenicity and antigenicity), increase thehydrodynamic size (size in solution) of the multipartite peptide whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic proteins.

The PEG molecules are typically characterized as having for example fromabout 2 to about 1000, or from about 2 to about 300 repeating units. Forexample water-soluble polymers, including but not limited to PEG,poly(ethylene oxide) (PEO), polyoxyethylene (POE), polyvinyl alcohols,hydroxyethyl celluloses, or dextrans, are commonly conjugated toproteins to increase stability or size, etc., of the protein asdescribed in U.S. Patent Publication No. 2012/0171115.

PEG, PEO and POE are oligomers or polymers of ethylene oxide. In thecase of PEG, these oligomers or polymers are produced by, e.g., anionicring opening polymerization of ethylene oxide initiated by nucleophilicattack of a hydroxide ion on the epoxide ring. One of the more usefulforms of PEG for protein modification is monomethoxy PEG (mPEG).

Preferred PEGs are monodisperse or polydisperse, preferablymonodisperse. The skilled artisan will be aware that PEG can bepolydisperse or monodisperse. Polydisperse PEG comprises a mixture ofPEGs having different molecular weights. In the case of polydispersePEGs, reference to a specific molecular weight will be understood torefer to the number average molecular weight of PEGs in the mixture. Thesize distribution is characterized statistically by its weight averagemolecular weight (MW) and its number average molecular weight (Mn), theratio of which is called the polydispersity index (Mw/Mn). MW and Mn aremeasured, in certain aspects, by mass spectroscopy. Most of thePEG-protein conjugates, particularly those conjugated to PEG larger than1 KD, exhibit a range of molecular weights due to a 30 polydispersenature of the parent PEG molecule. For example, in case of mPEG2K(Sunbright ME-020HS, NOF), actual molecular masses are distributed overa range of 1.5-3.0 KD with a polydispersity index of 1.036. Based on theforegoing, the skilled artisan will be aware that monodisperse PEGcomprises a mixture of PEGs comprising substantially the same molecularweight. Monodisperse PEGs are commercially available, e.g., fromPolypure AS, Norway.

The average or preferred molecular weight of the PEG may range from 500Da to 200 kDa, from 1 to 100 kDa, from 2 to 50 kDa, from 5 to 25 kDa, orfrom 5 kDa to 10 kDa, including any integers encompassed within theseranges.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For peptides or proteins, typical reactive aminoacids include lysine, cysteine, histidine, arginine, aspartic acid,glutamic acid, serine, threonine, tyrosine. The N-terminal amino groupand the C-terminal carboxylic acid can also be used as a site specificsite by conjugation with aldehyde functional polymers.

In certain embodiments, the PEG derivatives are produced by reacting thePEG polymer with a group that is reactive with hydroxyl groups,typically anhydrides, acid chlorides, chloroformates and carbonates. Inother embodiments, more efficient functional groups such as aldehyde,esters, amides, etc. are made available for protein conjugation.

In certain embodiments, heterobifunctional PEGs are used forconjugation. These heterobifunctional PEGs are useful for linking twoentities, where a hydrophilic, flexible and biocompatible spacer isneeded. Preferred end groups for heterobifunctional PEGs are maleimide,vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHSesters. In other embodiments, the pegylation agents contain branched, Yshaped or comb shaped polymers that show reduced viscosity and lack oforgan accumulation.

Various methods are known in the art for conjugating PEGs to peptides orproteins, as describe in U.S. Patent Publication No. 2012/0171115.Conjugation of PEGs may include the use of spacer moieties that arecleavable or non-cleavable. In some embodiments, the cleavable spacermoiety is a redox-cleavable spacer moiety, such that the spacer moietyis cleavable in environments with a lower redox potential, such thecytoplasm and other regions with higher concentrations of molecules withfree sulfhydryl groups. Examples of spacer moieties that may be cleaveddue to a change in redox potential include those containing disulfides.The cleaving stimulus can be provided upon intracellular uptake of theconjugated protein where the lower redox potential of the cytoplasmfacilitates cleavage of the spacer moiety. In the case of PEG, themolecule can be activated to facilitate its binding to amines orimidazoles, a carboxylic group, a hydroxyl group or a sulfhydryl group.

In another example, a decrease in pH causes cleavage of the spacer tothereby release of the compound into a target cell. A decrease in pH isimplicated in many physiological and pathological processes, such asendosome trafficking, tumour growth, inflammation, and myocardialischemia. The pH drops from a physiological 7.4 to 5-6 in endosomes or4-5 in lysosomes. Examples of acid sensitive spacer moieties which maybe used to target lysosomes or endosomes of cancer cells, include thosewith acid-cleavable bonds such as those found in acetals, ketals,orthoesters, hydrazones, trityls, cis-aconityls, or thiocarbamoyls (seefor example, U.S. Pat. Nos. 4,569,789, 4,631,190, 5,306,809, and5,665,358). Other exemplary acid-sensitive spacer moieties comprisedipeptide sequences Phe-Lys and Val-Lys.

Cleavable spacer moieties may be sensitive to biologically suppliedcleaving agents that are associated with a particular target cell, forexample, lysosomal or tumor-associated enzymes. Examples of linkingmoieties that can be cleaved enzymatically include, but are not limitedto, esters and endopeptidase cleavage recognition sites.

An activated PEG may be used with cyanuric chloride to produce a PEGdichlorotriazine derivative. This derivative can react with multiplefunctional nucleophilic functional groups, such as lysine, serine,tyrosine, cysteine and histidine. Two widely used forms of PEG used toconjugate to proteins are succinimidyl carbonate PEG and benzotriazolecarbonate PEG (BTC-PEG; U.S. Pat. No. 5,560,234). Both of thesecompounds react preferentially with lysine residues to form carbamatelinkages, however are also known to react with hystidine and tyrosine.SC-PEG is slightly more resistant to hydrolysis than BTC-PEG.

Another PEG useful for conjugating to proteins is PEG-propionaldehyde(U.S. Pat. No. 5,252,714). An advantage of this chemistry is that underacidic conditions (about pH5) it is largely selective for N-terminalα-amine thus avoiding potential problems with non-specific conjugation.An acetal derivative of PEG-propionaldehyde, i.e., PEG-acetaldehydeprovides an additional benefit in so far as it provides for longerstorage than PEG-propionaldehyde (U.S. Pat. No. 5,990,237).

Active esters of PEG carboxylic acids are probably one of the most usedacylating agents for protein conjugation. Active esters react withprimary amines near physiological conditions to form stable amides.Activation of PEG-carboxylic acids to succinimidyl active esters isaccomplished by reacting the PEG-carboxylic acid withN-hydroxysuccinimide (NHS or HOSu) and a carbodiimide. Exemplarycarboxylic acid derivatives of PEG include carboxymethylated PEG(CM-PEG), butanoic acid derivatives and propionic acid derivatives (U.S.Pat. No. 5,672,662). Changing the distance between the active ester andthe PEG backbone by the addition of methylene units can dramaticallyinfluence reactivity towards water and amines (e.g., by reducinghydrolysis). Alternatively or in addition, hydrolysis can be reduced byintroducing an .alpha.-branching moiety to the carboxylic acid.

PEGylation of free cysteine residues in a protein is useful forsite-specific conjugation (e.g., using a protein modified to includecysteine residues as described herein). Exemplary PEG derivatives forcysteine conjugation include PEG-maleimide, PEG-vinylsulfone,PEG-iodoacetamide and PEG-orthopyridyl disulfide. Exemplary methods forconjugating PEG to cysteine residues and for conjugation usingPEG-vinylsulfone are well known in the art.

U.S. Pat. No. 5,985,263 describes methods for conjugating PEG to thesecondary amine group of histidine, which has a lower pKa than theprimary amine. An advantage of this approach is that the acyl-histidinebond is not stable meaning that the peptide or protein is slowlyreleased (i.e., the conjugate behaves as a slow release formulation or apro-drug).

Another approach for PEGylation is to take advantage of a N-terminalserine or threonine, which can be converted to periodate as discussedabove. Using this approach, PEG has been conjugated to bioactiveproteins (e.g., Gaertner and Offord, 1996). PEG can also be conjugatedto carbohydrate groups.

The pharmaceutical composition of the present disclosure is formulatedto be compatible with its intended route of administration. Examples ofroutes of administration include parenteral, e.g., intrathecal,intra-arterial, intravenous, intradermal, subcutaneous, oral,transdermal (topical) and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine; propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfate; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating themultipartite peptide in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the multipartite peptide into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active peptide plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. The tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature,including a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Stertes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the peptides are delivered in the formof an aerosol spray from pressured container or dispenser which containsa suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forcontrolled or delayed release of the active ingredient. For example, incertain embodiments, the peptides may be delivered from with an entericcoating applied as a barrier to an oral formulation so as to preventrelease of the peptides before they reach the small intestine. As usedherein, the term “enteric coating” is a coating comprising of one ormore polymers having a pH dependent or pH-independent release profile.An enteric coated pill will not dissolve in the acidic juices of thestomach (pH ˜3), but they will in the alkaline (pH 7-9) environmentpresent in the small intestine or colon. An enteric polymer coatingtypically resists releases of the active agents until sometime after agastric emptying lag period of about 3-4 hours after administration.

Such enteric coatings or barrier coatings are also used to protectacid-unstable peptides from the stomach's acidic exposure, deliveringthem instead to a basic pH environment (intestine's pH 5.5 and above)where they do not degrade and can mediate their desired action. An oralformulation may comprise a plurality of barrier coatings comprising avariety of different materials to facilitate release in a temporalmanner. The coating may be a sugar coating, a film coating (e.g., basedon hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols, and/or polyvinylpyrrolidone)or a coating based on methacrylic acid copolymer, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac,and/or ethylcellulose. Furthermore, the formulation may additionallyinclude a time delay material such as glyceryl monostearate or glyceryldistearate.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from e.g. Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to tumor antigensor viral antigens) can also be used as pharmaceutically acceptablecarriers.

It is especially advantageous to formulate the peptide compositions indosage unit form for ease of administration and uniformity of dosage.Suitable unit dosage forms include, but are not limited to powders,tablets, pills, capsules, lozenges, suppositories, patches, nasalsprays, injectibles, implantable sustained-release formulations, lipidcomplexes, etc.

Methods of Treatment

In another aspect, the present disclosure provides methods for treatingcancers and infectious diseases. In some embodiments, the presentapplication relates to a method for treating cancer. The methodcomprises the step of administering to a subject in need thereof aneffective amount of a pharmaceutical composition comprising amultipartite peptide containing at least one secretion modifying region(SMR) peptide from HIV-1 Nef and at least one Clusterin (Clu)-bindingpeptide (Clu-BP). The subject may have a cancer selected from the groupconsisting of leukemia, lymphoma, carcinoma, melanoma, sarcoma, germcell tumor and blastoma.

As used herein, the term “leukemia” refers to progressive, malignantdiseases of the blood-forming organs and is generally characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow. Exemplary leukemias include,for example, acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “lymphoma” refers to a group of blood cell tumors that developfrom lymphocytes. Exemplary lymphomas include, for example, Hodgkin'slymphomas (HL) and the non-Hodgkin lymphomas.

The term “carcinoma” refers to the malignant growth of epithelial cellstending to infiltrate the surrounding tissues and give rise tometastases. Exemplary carcinomas include, for example, acinar carcinoma,acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare,basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolarcarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriformcarcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloidcarcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma,carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma,cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

The term “sarcoma” refers to a tumor made up of a substance like theembryonic connective tissue and is generally composed of closely packedcells embedded in a fibrillar or homogeneous substance. Exemplarysarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's Disease, multiplemyeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid,premalignant skin lesions, testicular cancer, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, and adrenal corticalcancer. In one embodiment, the subject has breast cancer.

In other embodiments, the present application relates to a method fortreating an infectious disease. The method comprises the step ofadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a multipartite peptide containingat least one secretion modifying region (SMR) peptide from HIV-1 Nef andat least one Clusterin (Clu)-binding peptide (Clu-BP).

In some embodiments, the infectious disease is caused by a bacterium. Insome embodiments, the infectious disease is caused by a fungus, In someembodiments, the infectious disease is caused by a parasite.

In some embodiments, the infectious disease is caused by a virusselected from the group consisting of human immunodeficiency virus type1 and type 2 (HIV-1 and HIV-2), human T-cell lymphotropic virus type Iand type II (HTLV-I and HTLV-II), hepatitis A virus, hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis Evirus (HEV), hepatitis G virus (HGV), parvovirus B19 virus, hepatitis Avirus, hepatitis G virus, hepatitis E virus, transfusion transmittedvirus (TTV), Epstein-Barr virus, human cytomegalovirus type 1 (HCMV-1),human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7),human herpesvirus type 8 (HHV-8), influenza type A viruses, includingsubtypes H1N1 and H5N1, human metapneumovirus, severe acute respiratorysyndrome (SARS) coronavirus, hantavirus, and RNA viruses fromArenaviridae (e.g., Lassa fever virus (LFV)), Pneumoviridae (e.g., humanmetapneumovirus), Filoviridae (e.g., Ebola virus (EBOV), Marburg virus(MBGV) and Zika virus); Bunyaviridae (e.g., Rift Valley fever virus(RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and hantavirus);Flaviviridae (West Nile virus (WNV), Dengue fever virus (DENV), yellowfever virus (YFV), GB virus C (GBV-C; formerly known as hepatitis Gvirus (HGV)); Rotaviridae (e.g., rotavirus), and combinations thereof.In one embodiment, the subject is infected with HIV-1 or HIV-2.

Combination Therapies

In certain embodiments, the SMR peptides of the present application arecombined with one or more additional anti-cancer agents. The anti-canceragent may be a mortalin (Hsp70) inhibitor; an alkylating agent; ananthracycline antibiotic; an anti-metabolite; a detoxifying agent; aninterferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; aHER2 inhibitor; a histone deacetylase inhibitor; a hormone oranti-hormonal agent; a mitotic inhibitor; aphosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; amammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor;a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathwayinhibitor; a centrosome declustering agent; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitor; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analogue orcombination thereof.

Exemplary mortalin (Hsp70) inhibitors include, but are not limited to,MKT-077(1-Ethyl-2-[[3-ethyl-5-(3-methyl-2(3H)-benzothiazolylidene)-4-oxo-2-thiazolidinylidene]methyl]-pyridiniumchloride), Omeprazole(5-Methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole),5-(N,N-Dimethyl)amiloride (DMA), 2-phenylethynesulfonamide (PES), JG-98(Li et al., ACS Med. Chem. Lett., (2013)4:1042-1047). Additionalmortalin inhibitors are described in U.S. Pat. No. 9,642,843 and U.S.Patent Publication Nos. 2012/0252818, 2017/0014434, and 2018/0002325.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary anthracycline antibiotics include, but are not limited to,doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone(Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine);daunorubicin liposomal (DaunoXome); dactinomycin (Cosmegen); epirubicin(Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin(Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodine131 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) and ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Exemplary histone deacetylase inhibitors include, but are not limitedto, vorinostat (Zolinza), valproic acid, romidepsin, entinostatabexinostat, givinostat, and mocetinostat.

Exemplary hormonal or anti-hormonal agents include, but are not limitedto, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol(Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur);fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA;Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex);bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone(Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera);abiraterone acetate (Zytiga); leuprorelin (Lupron); estramustine(Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix(Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone(Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary phosphatidyl-inositol-3 kinase (PI3K) inhibitors includewortmannin an irreversible inhibitor of PI3K, demethoxyviridin aderivative of wortmannin, LY294002, a reversible inhibitor of PI3K;BKM120 (Buparlisib); Idelalisib (a PI3K Delta inhibitor); duvelisib(IPI-145, an inhibitor of PI3K delta and gamma); alpelisib (BYL719), analpha-specific PI3K inhibitor; TGR 1202 (previously known as RP5264), anoral PI3K delta inhibitor; and copanlisib (BAY 80-6946), an inhibitorPI3Kα,δ isoforms predominantly.

Exemplary Akt inhibitors include, but are not limited to miltefosine,AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795,and SR13668.

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus;deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128,BEZ235, PI-103, Torin1, PP242, PP30, Ku-0063794, WAY-600, WYE-687,WYE-354, and CC-223.

Exemplary proteasomal inhibitors include, but are not limited to,bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib(CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912),marizomib (NPI-0052), and disufiram.

Exemplary PARP inhibitors include, but are not limited to, olaparib,iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016,MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338 andcombinations thereof.

Exemplary Ras/MAPK pathway inhibitors include, but are not limited to,trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026,R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, andGDC-0973.

Exemplary centrosome declustering agents include, but are not limitedto, griseofulvin; noscapine, noscapine derivatives, such as brominatednoscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN),N-(3-brormobenzyl) noscapine, aminonoscapine and water-solublederivatives thereof, CW069; the phenanthridene-derived poly(ADP-ribose)polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide,N2-(2-thienylmethyl)-5-nitro-2-furamide, andN2-benzyl-5-nitro-2-furamide.

Exemplary multi-kinase inhibitors include, but are not limited to,regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080;Zd6474; PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412;bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244);SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxel.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); and mimosine(Leucenol).

These additional chemotherapeutic agents may be loaded into liposomeswith the SMR peptides of the present application, in separate liposomalformulations co-administered with the SMR peptides, or by other modes ofadministration as otherwise employed (e.g., oral administration, i.v.injection etc.).

In certain particular embodiments, the SMR peptides of the presentapplication are combined with one or more mortalin inhibitors.

Dosages and Routes of Administration

Depending on the nature of the disease target, the multipartite peptidesof the present disclosure may be administered by any route, includingbut not limited to any of the various parenteral, gastrointestinal,inhalation, and topical (epicutaneous) routes of administration.Parenteral administration generally involves injections or infusions andincludes, for example, intravenous, intraarterial, intratumoral,intracardiac, intramuscular, intravesicular (e.g., to the bladder),intracerebral, intracerebroventricular, intraosseous infusion,intravitreal, intaarticular, intrathecal, epidural, intradermal,subcutaneous, transdermal, and intraperitoneal administration.Gastrointestinal administration includes oral, buccal, sublingual andrectal administration. The route of administration may involve local orsystemic delivery of the multipartite peptides.

As a general proposition, the therapeutically effective amount of themultipartite peptide administered will be in the range of about 1 ng/kgbody weight/day to about 100 mg/kg body weight/day whether by one ormore administrations. In a particular embodiment, the range ofmultipartite peptide administered is from about 1 ng/kg body weight/dayto about 1 μg/kg body weight/day, 1 ng/kg body weight/day to about 100ng/kg body weight/day, 1 ng/kg body weight/day to about 10 ng/kg bodyweight/day, 10 ng/kg body weight/day to about 1 μg/kg body weight/day,10 ng/kg body weight/day to about 100 ng/kg body weight/day, 100 ng/kgbody weight/day to about 1 μg/kg body weight/day, 100 ng/kg bodyweight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day toabout 10 μg/kg body weight/day, 1 μg/kg body weight/day to about 100μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 10 μg/kg body weight/day to about 1 mg/kg body weight/day,100 μg/kg body weight/day to about 10 mg/kg body weight/day, 1 mg/kgbody weight/day to about 100 mg/kg body weight/day and 10 mg/kg bodyweight/day to about 100 mg/kg body weight/day.

In other embodiments, the multipartite peptide is administered at adosage range of 1 ng-10 ng per injection, 10 ng-100 ng per injection,100 ng-1 μg per injection, 1 μg-10 μg per injection, 10 μg-100 μg perinjection, 100 μg-1 mg per injection, 1 mg-10 mg per injection, 10mg-100 mg per injection, and 100 mg-1000 mg per injection. Themultipartite peptide may be injected daily, or every 2, 3, 4, 5, 6 and 7days.

In other embodiments, the dose range of the multipartite peptideadministered is from about 1 ng/kg to about 100 mg/kg. In still anotherparticular embodiment, the range of antibody administered is from about1 ng/kg to about 10 ng/kg, about 10 ng/kg to about 100 ng/kg, about 100ng/kg to about 1 μg/kg, about 1 μg/kg to about 10 μg/kg, about 10 μg/kgto about 100 μg/kg, about 100 μg/kg to about 1 mg/kg, about 1 mg/kg toabout 10 mg/kg, about 10 mg/kg to about 100 mg/kg, about 0.5 mg/kg toabout 30 mg/kg, and about 1 mg/kg to about 15 mg/kg.

In other particular embodiments, the amount of multipartite peptideadministered is, or is about, 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03,0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300, 600 and 1000 mg/day.

The specific dose of multipartite peptide is determined by theparticular circumstances of the individual patient including the size,weight, age and sex of the patient, the nature and stage of the disease,the aggressiveness of the disease, and the route of administration ofthe pharmaceutical composition.

In certain embodiments, the multipartite peptide may be administered atleast once per day, typically once, twice, three times or four times perday with the doses given at equal intervals throughout the day and nightin order to maintain a constant presence of the drug in order to providesufficient efficacy. However, a skilled artisan will appreciate that atreatment schedule can be optimized for any given patient, and thatadministration of compound may occur less frequently than once per day.

Dosage unit form as used herein includes physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of the multipartite peptidecalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of the invention are dictated by and directly dependent onthe unique characteristics of the multipartite peptide and theparticular therapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the multipartite peptide of thepresent disclosure can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Peptidesexhibiting large therapeutic indices are preferred. While peptides thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such peptides to the site of affectedtissue in order to minimize potential damage to non-diseased cells and,thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch peptides lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any peptide usedin the methods of the present disclosure, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

When treating a cancer, any of the multipartite peptides of the presentdisclosure may be prescribed to be taken in combination with one or moreother anti-cancer agents. When used in such combination therapies, themultipartite peptides of the present disclosure and other pharmaceuticalagents may be administered simultaneously, by the same or differentroutes, or at different times during treatment. In particular, themultipartite peptides may be combined with a mortalin siRNA, ananti-cancer agent, such an alkylating agent; an anthracyclineantibiotic; an anti-metabolite; a detoxifying agent; an interferon; apolyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor;a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; aphosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; amammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor;a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathwayinhibitor; a centrosome declustering agent; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitor; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analogue,and combination thereof.

In some embodiments, the multipartite peptides of the present disclosureis administered in combination or concurrently with a chemotherapeuticagent, such as paclitaxel or cisplatin.

Likewise, when treating an infectious disease, the multipartite peptidesof the present disclosure may be prescribed to be taken in combinationwith one or more antiviral drugs. In certain embodiments, the antiviraldrug is a antiretroviral drug selected from the group consisting of:protease inhibitors, nucleoside reverse transcriptase inhibitors,nucleotide reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, integrase inhibitors, entry inhibitors, andmaturation inhibitors. Exemplary antiviral drugs include, but are notlimited to, abacavir, acyclovir, adefovir, amantadine, amdoxovir,amprenavir, antiprotease, apricitabine, arbidol, artemisinin,atazanafir, atripla, azidothymidine (AZT), bevirimat, boceprevir,butylated hydroxytoluene (BHT), cidofovir, combivir, darunavir,delavirdine, didanosine, dipivoxil, docosanol, edoxudine, efavirenz,elvitegravir, elvucitabine, emtricitabine, enfuviritide, entecavir,etravirine, famciclovir, foscarnet, fosamprenavir, gancyclovir,globoidnan A, GSK-572, HIV fusion inhibitors, hypericin, ibalizumab,idoxuridine, immunovir, indinavir, interferons (Types I, II and III),lamivudine, lersivirine, lopinivir, loviride, maraviroc, maribavir,MK-2048, molixan (NOV-205), moroxydine, nelfinavir, nevirapine, nexavir,non-nucleotide HIV RT inhibitors, oseltamivir, pegylated interferons(e.g., peginterferon alfa-2a), penciclovir, pencyclovir, peramivir,pleconaryl, podophyllotoxin, racivir, raltegravir, resquimod, ribavirin,rifampin, rilpivirine, rimantidine, ritonavir, saquinivir, stampidine,stavudine, taribavirin, tenofovir, tipranavir, trifluridine, trizivir,tromantidine, truvada, valaciclovir (Valtrex), valacyclovir,valganciclovir, vicriviroc, vidarabine, vivecon, zalcitabine, zanamivir(Relenza), zidovudine, and combinations thereof.

The treatment may be carried out for a period sufficient to achieve atherapeutic effect. Typically it is contemplated that treatment would becontinued indefinitely while the disease state persists, althoughdiscontinuation might be indicated if the pharmaceutical compositions nolonger produce a beneficial effect. The treating physician will know howto increase, decrease, or interrupt treatment based on patient response.

Production of the Multipartite Peptide

The multipartite peptides of the present disclosure can be chemicallysynthesized or produced from cells transformed with polynucleotideexpression vectors encoding the multipartite peptide. Multipartitepeptides of the present disclosure may be synthesized using traditionalliquid- or solid-phase synthesis. Fmoc and t-Boc solid phase peptidesynthesis (SPPS) can be employed to grow the peptides from carboxy toamino-terminus.

In other embodiments the multipartite peptides are synthesized usingrecombinant DNA technologies well known to those skilled in the art.Polynucleotide expression vectors can be designed to facilitatepreparative expression levels in many different cell hosts, includingbacteria, yeast, insect cells, and mammalian cells.

In one aspect, the present disclosure provides a host cell transformedwith a polynucleotide or expression vector encoding the multipartitepeptide. The host cells can be any bacterial or eukaryotic cell capableof expressing the multipartite peptide-encoding nucleic acids orexpression vectors described herein.

In another aspect, a method of producing a multipartite peptideaccording to the present disclosure comprises culturing a host celltransformed with a multipartite peptide-encoding polynucleotide orexpression vector under conditions that allows production of themultipartite peptide, and purifying the multipartite peptide from thecell. The peptides may be produced by culturing a cell transiently orstably expressing a multipartite peptide; and purifying the peptide fromthe cultured cells. Any cell capable of producing a functional peptidemay be used. The peptide-expressing cell may be of prokaryotic orbacterial origin, such as E. coli or it may be of eukaryotic ormammalian origin, such as a human cell. In other embodiments, the cellis a yeast cell or an insect cell. Where the cell is of eukaryoticorigin, the peptide-producing cell is preferably stably transformed witha polynucleotide so as to express the peptide.

The present disclosure is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

Example 1: Materials and Methods for Examples 2-8

1-1. Cell lines, reagents and antibodies. The MCF-7 cell line, anoninvasive estrogen receptor positive (ER+) and MDA-MB-231 cell line(ER negative) were purchased from the American Type Culture Collection(ATCC, Manassas, Va.). MCF-10A cell line, a non-tumorigenic epithelialcell line was also purchased from ATCC.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), Dulbecco'sModified Eagle's Medium (DMEM) with high glucose and FLUOROBRITE™ phenolred-free DMEM, (MCF-7) were purchased from Thermo Fisher Scientific(Rockford, Ill.) The RPMI 1640 medium (MDA-MB-231 cells) was obtainedfrom Life Technologies Company (Carlsbad, Calif.). The basal medium MEBMand the additive MEGM (MCF-10A cells) were obtained fromLonzal/Clonetics Corporation (Lonza, Walkersville, Md.). Paclitaxel waspurchased from Sellck-Chemon, (Houston, Tex.). Cisplatin was purchasedfrom EMD/Millipore (Billerica, Mass.). Annexin V-FITC/PI Apopto and PICell Cycle Kits were purchased from Nexcelom Bioscience (Lawrence,Mass.). The CD63 Rabbit polyclonal and Alix goat polyclonal antibodieswere purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.).The PEG-SMRwt-Clu PEG-SMRwt and PEG-SMRmut HIV-1 Nef peptides werepurchased from InnoPep Company (San Diego, Calif.).

1-2. Cell culture. Cells were cultured in the media described above withaddition of exosome-free fetal bovine serum (System Biosciences Inc.,Mountain View, Calif.), 100 units/mL penicillin, and 100 mg/mLstreptomycin and maintained in a humidified atmosphere at 37° C. and 5%CO₂.

1-3. Viability and proliferation. Human breast cancer cell lines wereseeded into 96-well plates (5000 cells/well) and treated for 24 hourswith various concentrations of SMR peptides including PEG-SMRwt-Clu,PEG-SMRwt and PEG-SMRmut to determine IC50 (inhibition concentration).Cell proliferation was determined using the MTT assay (MolecularDevices, Sunnyvale, Calif.). Control experiments were performed with MTTtreated cells alone and untreated cells, and on this basis, theincubation times of 24 hr and 48 hr were used for an MTT assay ofpeptide-treated cells (Stockerta JC. et al., 2012 and Riss TL. et al.,2015).

1-4. Cell cycle analysis. MCF-7 and MDA-MB-231 breast cancer cells werecultured into 6-well plates at 4×10⁵ cells per well and treated witheither paclitaxel and cisplatin or combined with PEG-SMRwt-CLU peptidefor 24 and 48 hours. Cell cycle analysis was performed using a propidiumiodide cell cycle assay and measured using a Cellometer (Nexcelom, MA).Further experiments were performed with SMR at IC50 concentration; theresults showed 1.12 μM of PEG-SMRwt-Clu, 0.28 μM of PEG-SMRwt on MCF-7cells for 24 hours and 0.28 μM of PEG-SMRwt-Clu, 0.42 μM of PEG-SMRwt onMDA-MB-231 cells for 24 hours for each cell time two stage. Breastcancer cells were seeded into 96-well plates at 5×10³ cells/ml, andtreated with either 1.6 μM//mL of paclitaxel, or 3 mg/mL cisplatin, 1.12μM/mL PEG-SMRwt-CLU peptide, SMR peptide combined with paclitaxel orwith cisplatin (MCF-7 cells). Alternatively 1.6 μM/mL paclitaxel or 2mg/mL cisplatin or 0.28 μM/mL of PEG-SMR-CLU peptide, or the peptidecombined with each of these drugs was used for MDA-MB-231 cells. Theconcentrations for cisplatin and paclitaxel were experimentallydetermined IC50 dosages for the different cell types (data not shown).At the end of the 24 hr or 48 hr incubations, the cells were assessed bythe Cellometry imaging cytometry assay.

All above steps were done on MCF-7 and MDA-MB-231 cells separately. Inorder to further understand whether this peptide functionssynergistically with chemotherapeutic drugs, 6 groups of cancer cellswere treated as follows: 1) untreated, 2) PEG-SMRwt-CLU, 3) paclitaxel,4) paclitaxel in combination with PEG-SMRwt-CLU, 5) cisplatin, 6)cisplatin in combination with PEG-SMRwt-CLU.

1-5. Assessment of apoptosis. Breast cancer cells were seeded into6-well plates at 4×10⁵ cells per well and treated with either paclitaxelor cisplatin or various concentrations of SMR peptides for 24 hours ordifferent time point. SMR as described above. Apoptosis was determinedthe using Annexin V-FITC detection kit (Nexcelom, MA) and visualized byCellometer imaging cytometry.

1-6. Exosome isolation and purification. Exosomes were isolated frombreast cancer cells by differential centrifugation as previouslydescribed (Ali SA. et al., 2010). Untreated tumor cells were used as acontrol. Briefly, the above treated and untreated cell culturesupernatants were centrifuged at 400×g for 10 minutes. The supernatantswere transferred to a clear tube and centrifuged at 10,000×g for 30minutes. The supernatants from the second spin were ultracentrifuged at200,000×g for 2 hours to pellet exosomes. Finally, the exosome pelletswere re-suspended with PBS and stored at 4° C. until used for analysis.

1-7. Exosome characterization by acetylcholinesterase (AchE) assay.Purified exosomes were quantitated by measurement of AchE as described(Ellman et al., 1961). Briefly, a 100 mM dithibionitrobenzoic (DTNB)solution was prepared for use as a stock color indicator, and a 28.9mg/mL acetylthiocholine iodide in PBS solution was prepared as a stocksubstrate. The stock substrate stock can be stored at −20° C. up to onemonth, while the color indicator can be stored at 4° C. for two weeks. Aworking solution was prepared by mixing 10 mL of PBS with 200 μL ofSubstrate and 500 μl of DTNB. 50 μL of each exosome sample wastransferred to 96 well microtitre plates, and a standard curve wasprepared using AchE from 0.98 mU/mL to 2000 mU/mL. After 50 μL ofstandards were added into separate wells, 200 μL of the working solutionwas added to all wells. After 20 min incubation, AchE activity wasmeasured at 450 nm using a SpectroMax M5 fluorimeter.

1-8. Exosome nanoparticle tracking analysis (NTA). Analysis of absolutesize distribution of exosomes was performed using NanoSight LM10 withNTA2.3 (NanoSight Ltd., Minton Park, UK). Particles were automaticallytracked and sized based on Brownian motion and the diffusioncoefficient. After isolation, the untreated and treated breast cancerexosomes were re-suspended in 0.5 mL of PBS. Control medium and filteredPBS were used as controls in this technique. The NTA measurementconditions were: temperature=21.0+/−0.5° C.; viscosity=0.99+/−0.01 cP;frames per second=25; measurement time=30 s. The detection threshold wassimilar in all samples. Two recordings were performed for each sample.

1-9. Western blot analysis. Exosomes were isolated from culturesupernatants as described above. Protein concentration was determined bymeasuring absorbance at 280 nm (Nanodrop 2000). Protein samples weredenatured in SDS-PAGE sample buffer by heating at 95° C. for 15 min.Criterion TGX Precast Gels (4-20% Bio-Rad, Richmond, Calif.) were usedto separate the proteins and blotted as previously described (Huang MB.et al 2004). Blots were incubated with the primary antibodies, anti-CD63and anti-Alix, followed by goat or rabbit anti-Ig secondary antibodies.Specific bands were detected using ECL chemiluminescent substrate (SantaCruz Biotechnology, Santa Cruz, Calif.) and visualized on the ImageQuantLAS 4000 imaging system (GE Healthcare, Piscataway, N.J. 08854).

1-10. Fluorescent N—Rh-PE measurement. The fluorescent phospholipidanalog N—Rh-PE [N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine] is a lipid marker of exosomes and intraluminal vesicles ofmultivesicular bodies as previously described (Willem J et al., 1990).Briefly, 10 mM of the N—Rh-PE was stored in chloroform/methanol (2:1). A5 μM N—Rh-PE solution in a pre-cooled reaction medium was then added tothe treated with MCF-7 breast cancer cells transfected withsiRNA-Negative or siRNA-HSPA9, and then were incubated at 4° C. for 1 h.After this incubation period, the medium was removed and the cells wereextensively washed with cold medium to remove excess unbound lipids.Labeled cells were cultured in complete RPMI-1640 with 10%exosome-depleted FBS medium heat inactivated at 37° C. overnight.Measurement of N—Rh-PE in the collected supernatants/exosomes wascarried out using a spectrometer at 550 nm and 590 nm excitation andemission wavelengths, respectively.

1-11. Transfection with mortalin antibody. MCF-7 breast cancer cellswere transfected with mortalin antibody using a Chariot kit (ActiveMotif, Carlsbad, Calif.) in accordance with the manufacturer's protocol.Following a 48 hour incubation of these cells, the exosomes wereisolated and measured via AchE assay and NanoSight analysis.

1-12. Transient transfection with small interfering RNA (siRNA). MCF-7breast cancer cells were transfected with double-stranded siRNAs usingAmaxa's Nucleofector kit (Lonza Walkersville Inc., Walkersville, Md.) inaccordance with the manufacturer's protocol. Transfection of plasmidswas carried out using Amaxa Biosystems Nucleofector II as recommended bythe supplier. Mortalin siRNAs were prepared as previously described(Shelton et al., J Virol(2012) 86(1): p. 406-19). Followingtransfection, the cells were incubated at 37° C. for 24, 48, 72 and 96hours, and exosomes were isolated and measured by AchE assay and Westernblotting.

1-13. Statistical analysis. Data was expressed as the mean standarddeviation (S.D.). A two-sample t-Test assuming equal variances was usedto compare the differences between controls and treated samples in eachgroup. A value of p≤0.05 was considered to be statistically significant.

Example 2: SMR Peptides Inhibit Cell Growth of Breast Cancer Cells

Breast cancer cells were treated for 24 hours with increasingconcentrations (35 nM/mL, 70 nM/mL, 140 nM/mL, 280 nM/mL, 560 nM/mL and1120 nM/mL) of PEG-SMRwt-Clu peptide in combination with eitherPEG-SMRwt or PEG-SMRmut peptides as controls. Both peptides containingthe SMRwt sequence inhibited breast cancer cell growth in adose-dependent manner (FIG. 1). For MCF-7 cells, 50% inhibition was seenwith 1.12 μM/mL of PEG-SMRwt-Clu and 0.28 μM/mL of PEG-SMRwt. ForMDA-MB-231 cells 50% inhibition was achieved with 0.28 μM/mL ofPEG-SMRwt-Clu and 0.42 μM/mL of PEG-SMRwt. The PEG-SMRmut peptide didnot inhibit proliferation.

Example 3: SMRwt Peptides Induce Cell Cycle Arrest in Breast CancerCells

The data indicated that PEG-SMRwt-CLU peptides induced cell cycle arrestin MCF-7 cells and MDA-MB-231 cells assayed at 48 hours (FIG. 2). Whencells were treated with the PEG-SMRwt-CLU peptide, or the peptidecombined with paclitaxel or cisplatin, they were blocked in G2/M phase,indicating that PEG-SMRwt-Clu peptides contribute to induction of G2/Marrest in breast cancer cells.

Example 4: SMRwt Peptides Increased the Sensitivity of Breast CancerCells to Cisplatin and Paclitaxel in MCF-7 Breast Cancer Cells

In a separate experiment, MCF-7 and MDA-MB-231 cells were treated witheither PEG-SMRwt-CLU or PEG-SMRmut-CLU alone, or in further combinationwith paclitaxel or cisplatin and then assayed for apoptosis by AnnexinV-FITC/PI assay. Both of these cell lines showed increased apoptosisrelative to the unmodified control peptides after the incubation withpaclitaxel and cisplatin for 48 hours (FIG. 3). Interestingly, thePEG-SMRwt-Clu peptide increased the level of drug-induced apoptosis inMCF-7 cells, but not in MDA-MB-231 cells.

Example 5: SMR Peptides Block Exosome Release in Breast Cancer Cells

Acetylcholinesterase (AchE) assays, NanoSight analysis and Western blotanalysis were performed to characterize exosomes released from MCF-7 andMDA-MB-231 human breast cancer cells treated for 48 hr with the variouspeptides. The results indicated that exosome release was inhibited bythe SMRwt peptides.

AchE activity in exosomes was assayed and the results of this analysisare shown in FIGS. 4A and 4B. In MCF-7 cells, the control exosomes werefound to contain 113.49 mU/mL of AchE activity. In contrast, 41.95 mU/mLof activity was found in cells treated with PEG-SMRwt-CLU peptide; 51.87mU/mL activity was found in cells treated with PEG-SMRwt-CLU incombination with paclitaxel; and 16.95 mU/mL activity was found in cellstreated with PEG-SMRwt-CLU in combination with (FIG. 4A). In MDA-MB-231cells, the control exosomes contained 118.48 mU/mL of AchE activity,whereas 66.77 mU/mL activity was found in cells treated withPEG-SMRwt-CLU peptide; 64.15 mU/mL activity was found in cells treatedwith PEG-SMRwt-CLU peptide in combination with paclitaxel; and 27.0mU/mL activity was found in cells treated with PEG-SMRwt-CLU incombination with cisplatin (FIG. 4B).

Analysis of exosomes concentration and size distribution was assayed byNanoSight LM10 Nanoparticle Tracking Analysis (NTA). With NTA, particlesare automatically tracked and sized based on Brownian motion and theassociated diffusion coefficient. Before analysis of the samples by NTA,it was determined that salt aggregates from the PBS did not contributeto background and the equipment was free of contaminant particles. Theuntreated MCF-7 cell control medium showed a considerable number ofparticles (5.16×10⁹ particles/ml) (FIG. 4, Panel C). However, a reducednumber of particles was found in MCF-7 cells treated with PEG-SMRwt-CLU(3.28×10⁸ particles/ml, p<2.40E-06), PEG-SMRwt-CLU in combination withpaclitaxel (5.7×10′ particles/mL, p<0.0008) and PEG-SMRwt-CLU incombination with cisplatin (3.77×10⁸ particles/mL, p<0.0001) (FIG. 4,Panel C).

Similarly, whereas control media from MDA-MB-231 cultures also showed aconsiderable number of particles (4.7×10⁹ particles/ml), a reducednumber of particles was found in MDA-MB-231 cells treated withPEG-SMRwt-CLU peptide (6.8×10⁸ particles/ml, p<3.96E-05), PEG-SMRwt-CLUpeptide in combination with paclitaxel (7.5×10^(∧)8 particles/mL,p<0.001) and PEG-SMRwt-CLU peptide in combination with cisplatin(3.06×10⁸ particles/mL, p<5.37E-05) (FIG. 4, Panel D). By NTA analysis,the size of the exosomes was estimated to range between 30 to 47 nm inboth breast cancer cell lines.

Finally, Western blot analysis was used to detect exosome proteins incontrol- and peptide-treated cultures. The results of this analysisrevealed the presence of human CD63 and Alix markers in the all exosomesisolated from MCF-7 cells (FIG. 5) and MDA-MB-231 cells (FIG. 6).Control exosomes showed higher expression of human CD63 from MCF-7 cellsand higher expression of Alix from MDA-MB-231 cells.

Example 6: Blocking the SMR-Mortalin Interaction Blocks Exosome Releasein Breast Cancer Cells

A previous study identified the HSP70 family protein, mortalin (encodedby HSPA9) as a binding partner for HIV-1 Nef SMR, and showed thatdisruption of HIV-1 Nef SMR-mortalin binding interfered with exosomerelease (Shelton et al., J Virol (2012) 86(1): p. 406-19). To testwhether an analogous interaction accounts for the observed PEG-SMRwt-CLUeffect on exosome release from breast cancer cells, MCF-7 cells weretransfected with antibody to mortalin or antibody to α-tubulin as acontrol. The anti-mortalin treated cells were found to be significantlyimpaired in exosome release as measured by AchE assay (FIG. 7, Panel A)and slightly less so when measured by NTA assay (FIG. 7, Panel B). Theeffect of treatment with anti-mortalin was similar to the effect oftreating MCF-7 cells with PEG-SMRwt-CLU peptide.

To further validate the significance of this mortalin-mediated processin cancer cells, expression of mortalin protein was knocked down bytransfecting MCF-7 cells with a plasmid construct expressing a mortalinsiRNA. The mortalin siRNAs were found to block exosome secretion asevidenced by AchE assay and membrane fluorescence (N—Rh-PE) assays atall time points tested (FIG. 8, Panels A and B) in the absence of anycell toxicity (FIG. 8, Panel C). The exosomes from siRNA-transfectedcells were further assayed for expression of mortalin and the exosomemarker CD63, a tetraspanins by Western blot analysis. The results ofthis analysis showed that expression of both mortalin and CD63 wassignificantly decreased at 48 h on through to 96 h (FIG. 8, Panels D andE).

Example 7: Materials and Methods for Experiments in Examples 8-14

7-1. Cell Lines, sera, chemicals and antibodies. MDA-MB-231, MCF-7,MCF-10A and K-562 cells were purchased from American Type CultureCollection (ATCC, Manassas, Va.). Cells were cultured in RPMI 1640(Thermo Fisher Scientific, Rockford, Ill.) supplemented with 10% heatinactivated FBS (MedSupply Partners (Atlanta, Ga.)), 1% glutamine, 100mg/ml penicillin, and 100 mg/ml streptomycin (Life Technologies,Carlsbad, Calif.) at 37° C. and 5% CO₂. Normal human serum (NHS)(MedSupply Partners (Atlanta, Ga.)) was used as the source forcomplement proteins. Heat-inactivated normal human serum (NIS) wasprepared by heating serum at 56° C. for 45 min. Rabbit polyclonalanti-mortalin antibody (anti-Grp75) was purchased from Abcam, Inc.,(Cambridge, Mass.); goat polyclonal anti-Alix antibody and mousemonoclonal anti-α-Tubulin antibody were purchased from Sigma-Aldrich,Inc., (Louis, Mo.). Peroxidase-conjugated goat anti-mouse IgG,peroxidase conjugated rabbit anti-goat IgG, and FITC-conjugated goatanti-mouse IgG were purchased from Thermo Fisher Scientific, Inc.,(Rockford, Ill.).

7-2. Peptides. The PEG-SMRwt-Clu, PEG-SMRmut-Clu, SMRwt-CPP andSMRmut-CPP peptides were custom made by InnoPep Inc. (San Diego, Calif.)(FIG. 9).

7-3. Mortalin (HSPA9) DNA constructs. The BLOCK-iT Pol II miR RNAiexpression vector kit from Life Technologies Corporation (Carlsbad,Calif.) and the mortalin (HSPA9) primers Hmi 408224 to Hmi 408227 wereused to generate an expression vector with spectinomycin resistance(miR-mortalin) that expresses mortalin microRNA (miRNA; miR). The kitalso contained a corresponding pcDNA6.2-GW/miR-negative control plasmid(miR-neg) predicted not to target any known vertebrate gene.

7-4. Cell proliferation and cytotoxicity assay. Human breast cancer celllines MCF-7, MDAMB-231, and leukemia K562 cells, and non-tumorigenicMCF-10A cells are seeded into 96-well plates (5000 cells/well) andtreated for 24 hours with 0-1120 nM SMRwt-CPP or SMRmut-CPP (FIG. 10).Cell proliferation was determined using the MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye assayby SpectraMax M5 Fluorescence Plate Reader (Molecular Devices.Sunnyvale, Calif.). Cell viability was estimated by the conversion ofyellow MTT by mitochondrial dehydrogenases of living cells to purpleformazon (MTT assay). Control experiments were performed with cellstreated or untreated with MTT, and on this basis, an incubation time of24 hours was used for MTT assays of peptide-treated cells (Huang, M. B.,et al., Oncotarget, 2017. 8(7): pp. 11302-11315.). Statisticalsignificance of results was determined from three independentexperiments including triplet or quadruplet sets in each experiment.

7-5. Exosome isolation and purification. Exosomes were isolated frombreast cancer cells and leukemia cells using the ExoQuick-TC ExosomePrecipitation Kit (System Biosciencences [SBI], Mountain View Calif.) orthe miRCURY Exosome Isolation Kit (EXIQON, Woburn, Mass.) according tothe manufacturer's instructions. Untreated tumor cells were used as acontrol. Briefly, treated and untreated cell culture supernatants (10mL) were centrifuged at 3000×g for 15 minutes. The resultingsupernatants were transferred to a clear tube, 2 mL of ExoQuick-TCbuffer was added, mixed and incubated overnight at 4° C. Following theincubation, the sample contents were centrifuged at 1500×g for 30minutes, the supernatants were aspirated off, the pellets werecentrifuged at 1500×g for 5 minutes and any further traces ofsupernatant were removed (SBI). Alternatively, treated and untreatedcell culture supernatants were centrifuged at 10,000×g for 5 minutes toremove cell debris, supernatants (10 ml) were transferred into newtubes, and 4 mL of precipitation buffer (EXIQON) was added, and thecontents were mixed and incubated at 4° C. overnight. Following theincubation, the sample contents were centrifuged at 10,000×g for 30minutes (EXIQON). Exosome-containing pellets were re-suspended in PBSand stored at 4° C. or −80° C.

7-6. Exosome characterization by acetylcholinesterase (AchE) assay.Purified exosomes were quantitated by measurement of AchE as previouslydescribed (Huang et al., Oncotarget, 2017. 8(7): pp. 11302-11315).Briefly, 100 mM dithibionitrobenzoic (DTNB) was prepared as a stockcolor indicator, and prepared a stock substrate containing 28.9 mg/mL ofacetylthiocholine iodide in PBS. Substrate stock can be stored at −20°C. up to one month; the stock color indicator can be stored at 4° C. fortwo weeks. A working solution was prepared by mixing 10 mL of PBS with200 μL of substrate and 500 μl of DTNB. 50 μL of each exosome sample wastransferred to a 96 well microtitre plate, and a standard curve for AchEconcentrations was prepared using AchE from 0.98 mU/mL to 2000 mU/mL.After 50 μL of standards were added into separate wells, 200 μL of theworking solution was added to each well. After a 20 min incubation, AchEactivity was measured at 450 nm using a SpectroMax M5 fluorometer (FIG.11).

7-7. Protein analysis by Western blotting. Exosomes were isolated fromcell culture supernatants as described above. Protein concentrationswere determined by measuring absorbance at 280 nm (Nanodrop 2000).Cultured cells were treated with PEG-SMR-Clu, SMR-CPP peptides, MKT-077,Omeprazole or DMA at 37° C. for 48 hr. To evaluate mortalin, vimentinand exosome expression, Western blot analysis was performed usinganti-mortalin (Grp-75), anti-Vimentin and anti-Alix antibodies,respectively (FIG. 12).

For collection of proteins secreted from cells undergoing complementattack, cells were treated with an anti-CXCR4 antibody and normal humanserum or heat-inactivated human serum for 10 min at 37° C. The cellswere then washed with PBS and suspended in medium and incubated at 37°C. After 20 min, the cells were removed by centrifugation at 350×g for 5min (FIG. 15).

Exosomes and cell lysates were prepared by incubating 5-10 min at 95° C.in sample buffer. The lysates were subjected to SDS-PAGE under reducingconditions (150 mM DTT) in 4-20% Criterion™ TGX™ precast gels (BioRad,Hercules, Calif.) and then transferred onto a nitrocellulose membrane(Bio-Rad, Hercules, Calif.). Nitrocellulose membranes were blocked with5% skim milk (MedSupply, Atlanta, Ga.) in TBS containing 0.05% Tween 20(TBST) for 1 h at room temperature. The membranes were treated withanti-mortalin (Grp-75) mAb or anti-α-tubulin mAb for cell lysates (FIG.15, Panels A and B) or with anti-Alix and anti-vimentin or anti-mortalinfor exosomes (FIG. 12), followed by peroxidase-conjugated goatanti-mouse IgG. Bands were developed with an ECL chemiluminescentsubstrate (Santa Cruz Biotechnology, Santa Cruz, Calif.) and exposed toan ImageQuant LAS 4000 imaging system (GE Healthcare, Piscataway, N.J.08854).

7-8. Apoptosis assay by flow cytometry. For assessment of apoptosis,MCF-7 and MDA-MB-231 breast cancer cells and were seeded into 6-wellplates at 4×10⁵ cells per well and treated for 48 hours with 925 nM/mLof MKT-077 on MDA-MB-231 cells, 500 nM/mL of MKT-077 on MCF-7 cells,337.5 nM/mL of MKT-077 on K562 cells, 300 μM/mL of DMA, 200 μM/mL ofOmeprazole, 1.6 uM/mL of paclitaxel, 2 mg/mL of cisplatin, or combinedtreatment with either paclitaxel or cisplatin in combination with eachof the three mortalin inhibitors. Apoptosis was determined using theTUNEL detection kit (Nexcelom, MA) and GUAVA easyCyte HT (EMD MilliporeCorporation, Temecula, Calif.) system for fluorometric detection. (FIG.13).

7-9. Complement-mediated cell cytotoxicity assay. Complement-mediatedcell cytotoxicity assays were performed as previously described (Reiteret al., Mol Immunol. (1992) 29(6): p. 771-81). Briefly, MCF-7,MDA-MB-231 and K562 cells were either mock-transfected or transfectedwith mortalin siRNA. Cells were incubated with 1 μg/ml of anti-CXCR4antibody for 30 min at 4° C., followed by treatment with PEG-SMRwt-Clupeptide or PEG-SMRmut-Clu peptide or SMRwt-CPP peptide or SMRmut-CPPpeptide alone, or treated with SMR peptide combined with complement fromnormal human serum (NHS) or heat-inactivated normal human serum (NIS)for 60 min at 37° C. Percentage of cells lysed were determined by atrypan blue exclusion assay using the TC10™ Automated Cell Counter(Bio-Rad, Richmond Calif.). (FIG. 14, FIG. 15, Panel D).

7-10. Transient transfection with mortalin small interfering RNA(siRNA). MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cellswere plated in T-75 flasks with exosome-free medium. After 24 hours in a37° C. incubator, the cells were transfected with a vector expressingdouble-stranded mortalin siRNAs (see section 7-3) using Amaxa'sNucleofector II kit (Lonza Walkersville Inc., Walkersville, Md.)according to the manufacturer's protocol. Mock-transfected cells(“Mock”) or cells treated a non-mortalin siRNA expression vector (Neg)were used as negative controls. Following transfection, the cells wereincubated in culture medium at 37° C. for 72 hours before beingevaluated by Western blot analysis (FIG. 15, Panel A) and an AchE assay(FIG. 15, Panel C).

7-11. Exosome nanoparticle tracking analysis (NTA). Analysis of absolutesize distribution of exosomes (300 μL) was performed using NanoSightLM10 with NTA2.3 (NanoSight Ltd., Minton Park, UK). Particles wereautomatically tracked and sized based on Brownian motion and thediffusion coefficient. After isolation, the untreated and treated breastcancer cell or leukemia cell exosomes were re-suspended in 0.5 mL ofPBS. Control medium and filtered PBS were used as controls in thistechnique. The NTA measurement conditions were: temperature=21.0+/−0.5°C.; viscosity=0.99+/−0.01 cP; frames per second=25; measurement time=30s. The detection threshold was similar in all samples. Two recordingswere performed for each sample (FIG. 15, Panel C).

7-12. Cell migration assay. Cell migration assays were performed usingthe InnoCyte Cell Migration Assay (EMD Millipore Corporation, Temecula,Calif.) according to the manufacturer's instructions. Briefly, cellmigration was monitored in the presence and absence of SMRwt peptides orlatrunculin A (positive control inhibitor), which were added to thelower chamber of a 96-well plate. MCF-7 and MDA-MB-231 breast cancercells and non-tumorigenic MCF-10A breast cells were harvested by 0.25%Trypsin-EDTA (Life Technologies) release and resuspended at250,000500,000 cells/ml in serum-free RPMI 1640 media. A total of25,000-50,000 cells was added to the upper chamber and allowed tomigrate through an 8-μm pore-size membrane for 24 hours at 37° C. in a5% CO₂ atmosphere. Cells that migrated through the membrane weredetached and labeled with Calcein-AM fluorescent dye. Fluorescence wasmeasured using a Fluorescence Plate Reader (Molecular Devices,Sunnyvale, Calif.) with an excitation wavelength of 485 nm and anemission wavelength of 515 nm. The experiment was repeated intriplicate, and each conditioned experiment was performed inquadruplicate.

13. Statistical analysis. Data are expressed as the mean standarddeviation (S.D.). A two-sample t-test assuming equal variances was usedto compare the differences between controls and treated samples in eachgroup. A value of p≤0.05 was statistically significant.

Example 8: SMRwt-CPP Peptides Inhibit Cell Proliferation and Growth inBreast and Leukemia Cancer Cell Lines

MCF-7 and MDA-MB-231 breast cancer cells and K562 leukemia cell cultureswere treated for 24 hours with increasing concentrations (35 nM/mL, 70nM/mL, 140 nM/mL, 280 nM/mL, 560 nM/mL and 1120 nM/mL) of eitherSMRwt-CPP peptide or the negative control peptide SMRmut-CPP. As shownin FIG. 10, growth was inhibited by the in all cells tested in adose-dependent manner by SMRwt-CPP peptide, but not the negative controlpeptide. The inhibition observed was found to be dose dependent, with atypical sigmoidal shape of dose response, corroborating validity of theobservations. The growth curves further relate the specific dosageproviding 50% inhibition (IC50) by SMRwt-CPP, whereby the IC50 againstMCF-7 was 180 nM/mL (FIG. 10, Panel A), against MDA-MB-231 was 476 nM/mL(FIG. 10, Panel B) and against K562 was 907 nM/mL (FIG. 10, Panel D).Non-tumorigenic MCF-10A breast cells were used as a negative cellcontrol and were not affected by SMRwt-CPP (FIG. 10, Panel C).

Example 9: SMRwt Peptides and Mortalin Inhibitors Block Exosome Releasein Breast and Leukemia Cancer Cell Lines

PEG-SMRwt-Clu peptide was previously shown to block exosome release inbreast cancer cells (Huang et al., Oncotarget (2017) 8(7): p.11302-11315). To further examine the underlying mechanism for blockingexosome release, a study was extended to examine the effects of theSMRwt peptides, PEG-SMRwt-Clu peptide and SMRwt-CPP peptide, and themortalin inhibitors MKT-077, Omeprazole, and DMA on exosome release. Inparticular, acetylcholinesterase (AchE) assays, NanoSight analysis andWestern blot analysis were performed to characterize exosomes releasedfrom MCF-7 and MDA-MB-231 human breast cancer cells and K562 leukemiacells. Cells were treated for five days at 37° C. with the variouspeptides and mortalin inhibitors as indicated in FIG. 11. Panels A, Band C depict the relative numbers of exosomes released as a function oftime as measured by an acetylcholinesterase (AchE) assay in MCF-7 breastcancers cells (FIG. 11, Panel A); MDA-MB-231 breast cancer cells (FIG.11, Panel B); and K562 leukemia cells (FIG. 11, Panel C).

The results of this analysis show that the SMRwt peptides, PEG-SMRwt-Clupeptide (SEQ ID NO: 37) and SMRwt-CPP peptide (SEQ ID NO: 39), and themortalin inhibitors, MKT-077, Omeprazole and DMA all reduced the numberof exosomes release over the entire five day treatment period. As shownin FIG. 11, Panels A-C, there was an initial increase in exosome releaseon days 1 and/or 2 under all treatment conditions and a subsequentdecrease in exosome release thereafter, although the initial increasesand subsequent decreases were substantially reduced in cells treatedwith the SMRwt peptides or mortalin inhibitors. In particular, AchEconcentrations in MCF-7 cells treated with the negative controls (i.e.,mock, PEG-SMRmut-Clu (SEQ ID NO: 43), and SMRmut-CPP (SEQ ID NO: 41))were 155.13 mU/mL, 151.5 mU/mL and 150.06 mU/mL, respectively. On day 3,a rapid decrease was observed under these treatment conditions (69.15mU/mL, 69.0 mU/mL and 66.05 mU/mL) with a subsequent increase in AChEconcentrations on day 4 and day 5.

In contrast, MCF-7 cells treated with PEG-SMRwt-Clu (SEQ ID NO: 37),SMRwt-CPP (SEQ ID NO: 39), MKT-077, Omeprazole, and DMA were found toexhibit reduced AChE concentrations throughout days 1 to day 5, whereinthe average AChE concentrations were 31 mU/mL, 22 mU/mL, 22 mU/mL 26mU/mL and 26 mU/mL, respectively.

FIG. 11, Panel B shows the results obtained for similar treatments inMDA-MB-231 breast cancer cells. In this case, initial increases in AChEof 146.52 mU/mL, 146.79 mU/mL and 141.47 mU/mL were observed on day 1for mock, PEG-SMRmut-Clu, and SMRmut-CPP treatments, respectively, whichwere followed by decreases in AChE concentrations on days 2-5. InMDA-MB-231 cells treated with PEG-SMRwt-Clu, SMRwt-CPP, MKT-077,Omeprazole, and DMA, however, the AChE levels were maintained at averageconcentration levels throughout day 1 to 5 of 42 mU/mL, 29 mU/mL, 29mU/mL, 25 mU/mL and 37 mU/mL, respectively.

FIG. 11, Panel C shows the results obtained for similar treatments inK562 leukemia cells. In this case, average AChE levels in the negativecontrol groups from days 1 to 5 were 48 mU/mL, 71 mU/mL, 75 mU/mL, 69mU/mL and 59 mU/mL, respectively. By contrast, average AChE levels ondays 1-3 and 5 in K562 cells treated with PEG-SMRwt-Clu, SMRwt-CPP,MKT-077, Omeprazole, and DMA were 18 mU/mL, 25 mU/mL, 21 mU/mL and 18mU/mL with day 4 showing an increase relative to the other days (i.e.,41 mU/mL). These results further confirm that exosome release wasinhibited by all experimental treatments (FIG. 11).

Example 10: Effect of SMRwt Peptides and Mortalin Inhibitors on ExosomalProtein Content

Western blot analysis was used to examine the effects of the SMRwtpeptides, PEG-SMRwt-Clu peptide and SMRwt-CPP peptide, and the mortalininhibitors MKT-077, Omeprazole, and DMA on exosomal protein content inMCF-7, MDA-MB-231 and K562 cells. Negative control treatments includedPEG-SMRmut-Clu peptide and SMRmut-CPP peptide. Exosomes were isolatedfrom all treated and untreated cell lines screened. Western blotanalyses were carried out to evaluate the expression levels of theexosomal proteins, Alix, mortalin, and vimentin in MCF-7 breast cancercells (FIG. 12, Panel A, left sub-panel), MDA-MB-231 breast cancer cells(FIG. 12, Panel A, middle sub-panel), and in K562 leukemia cells (FIG.12, Panel A, right sub-panel).

As shown in FIG. 12, Panel A, left sub-panel and FIG. 12, Panel B, topleft sub-panel), Alix expression in exosomes from MCF-7 cells weresignificantly decreased in PEG-SMRwt-CLU, SMRwt-CPP and MKT-077treatments. However, as shown in FIG. 12, Panel B, top left sub-panel,treatment of MCF-7 cells with Omeprazole or DMA were not accompanied bysignificant reductions in Alix expression. Vimentin expression inexosomes from MCF-7 cells was significantly decreased for all treatments(PEG-SMRwt-Clu, SMRwt-CPP, MKT-077, omeprazole and DMA) relative to thenegative controls (PEG-SMRmut-Clu and SMRmut-CPP)(FIG. 12, Panel B,middle left sub-panel. Mortalin expression in MCF-7 exosomes was onlysignificantly decreased following treatment with PEG-SMRwt-Clu peptideor MKT-077, however, no significant changes in mortalin expression wereobserved following treatment with SMRwt-CPP, Omeprazole, or DMA (FIG.12, Panel B, bottom left sub-panel).

In MDA-MB-231 breast cancer cells (FIG. 12, Panel A, middle topsub-panel and FIG. 12, Panel B, middle top sub-panel), Alix expressionin MDA-MB-231 exosomes was found to be significantly decreased in cellstreated with PEG-SMRwt-Clu and SMRwt-CPP, however, no significantchanges were observed following treatment with the three mortalininhibitors. Vimentin expression was significantly decreased followingtreatment with PEG-SMRwt-Clu, SMRwt-CPP and DMA, however, no significantchange was observed following treatment with MKT-077 or omeprazole (FIG.12, Panel B, middle top sub-panel). Mortalin expression wassignificantly decreased following treatment with PEG-SMRwt-Clu andSMRwt-CPP treatment, and to a lesser extent following DMA treatment(FIG. 12, Panel B, middle bottom sub-panel. No significant change wasobserved following treatment of MDA-MB-231 cells with MKT-077 oromeprazole treatment.

In K562 leukemia cells (FIG. 12 Panel A, top right sub-panel and FIG. 12Panel B, top right sub-panel), Alix expression in K562 exosomes wassignificantly decreased following treatment with PEG-SMRwt-Clu,SMRwt-CPP, MKT-077, omeprazole and DMA. In contrast, vimentin expressionin K562 exosomes was significantly following treatment with MKT-077,Omeprazole and DMA, however, no significant changes in vimentinexpression were observed following treatment with any of the wild-typeor mutant SMR peptides (FIG. 12, Panel B, middle right sub-panel).Mortalin expression was significantly decreased following treatment withPEG-SMRwt-Clu, SMRwt-CPP, MKT-077, omeprazole and DMA. Taken together,the data from FIGS. 11 and 12 suggest that exosome numbers and exosomeprotein contents are modulated differently depending on the specifictreatment.

Example 11: Effect of Mortalin Inhibitors on Paclitaxel- andCisplatin-Induced Apoptosis

The effect of mortalin inhibitor drugs (MKT-077, DMA, and Omeprazole)were examined alone and in combination with paclitaxel (or cisplatin) toobserve their effects on apoptosis in MBA-MD-231, MCF-7, and K562 cells.In particular, these cells were treated for 48 hours with 300 μM/mL ofDMA, 200 μM/mL of Omeprazole, or different concentrations of MKT-077depending on the cell (MDA-MB-231, 925 nM/mL; MCF-7, 500 nM/mL, K562cells, 337.5 nM/mL), each treatment being conducted alone or incombination with 1.6 uM/mL of paclitaxel or 2 mg/mL of cisplatin andassayed for apoptosis by a TUNEL assay (FIG. 13).

The results showed decreased apoptosis relative (i) to the MKT-077 alone(52.13%) versus incubation with paclitaxel (21.14%) and cisplatin(27.53%) on MDA-MB-231 cells (FIG. 13, Panel A). Also, decreasedapoptosis relative to the MKT-077 alone (90.1%) versus with cisplatin(77.13%) on MCF-7 cells (FIG. 13, Panel B). And decreased apoptosisrelative to the MKT-077 alone (82.7%) versus with cisplatin (60.43%) onK562 cells (FIG. 13, Panel C). However, no significant changes wereobserved with paclitaxel on MCF-7 cells and K562 cells. (ii) Decreasedapoptosis with DMA alone (30.43%) versus incubated with cisplatin(23.03%) on MDA-MB-231 cells (FIG. 13, Panel A); decreased apoptosisrelative to DMA alone (11.83%) versus with paclitaxel (9.43%) on MCF-7cells (FIG. 13, Panel B); and decreased apoptosis relative to the DMAalone (15.2%) versus with paclitaxel (1.63%) and cisplatin (11.7%) onK562 cells (FIG. 13, Panel C). No significant changes were observed withpaclitaxel on MCF-7 cells, and with cisplatin in MCF-7 cells. (iii)Increased apoptosis was observed for omeprazole alone (3.0%) versusincubated with paclitaxel (15.33%) and cisplatin (29.05%) on MDA-MB-231cells (FIG. 13, Panel A); increased apoptosis for omeprazole alone(1.1%) versus with cisplatin (29.05%) on MCF-7 cells (FIG. 13, Panel B);and increased apoptosis relative to the omeprazole alone (6.1%) versuswith cisplatin (13.5%) on K562 cells (FIG. 13, Panel C). No significantchanges were observed with paclitaxel on MCF-7 cells and K562 cellsalthough a very small, synergistic effect of omeprazole in combinationwith either drug was observed.

Example 12: SMRwt Peptides Block Mortalin-Driven Exosome Release ofComplement-Dependent Cytotoxicity

Complement-mediated cytotoxicity is a normal cellular mechanism forridding the host of tumor cells. Mortalin/GRP75 has been shown to bindcomplement factor C9 and play a major role in development of resistanceto complement-dependent cytotoxicity via mortalin induced exocytosis ofthe membrane attack complex (MAC) via exosomes (Pilzer et al., Intl.Immunol., (2005) 17(9): p. 1239-1248). Therefore, it was of interest tosee whether SMR peptide driven mortalin sequestration and functionaldisruption increases cell sensitivity to complement-induced cell death.Accordingly, MCF-7, MDA-MB-231, and K562 cells were treated with 280 nMPEG-SMRwt-Clu peptide or 560 nM SMRwt-CPP peptide alone for 60 min at37° C., followed by treatment with 1 μg/mL anti-CXCR4 antibody and 50 μLnormal human serum (NHS; as a source of complement) or normalheat-inactivated (human) serum (NIS) for an additional 60 min at 37° C.(FIG. 14). PEG-SMRmut-Clu peptide and SMRmut-CPP peptide were used asnegative controls. The percentage of stained (dead) cells was determinedby trypan blue exclusion and is representative of 3 independentexperiments.

In the presence of functional complement (NHS), both of the SMRwtpeptides significantly induced tumor cell death. More specifically, inMCF-7 cells treated with PEG-SMRwt-Clu, cell death increased from 2.1%under complement-negative conditions (bar 4) to 26% undercomplement-positive conditions (bar 8)(FIG. 14, Panel A); likewise, inMCF-7 cells treated with SMRwt-CPP, cell death increased from 2.1% undercomplement-negative conditions (bar 6) to 29% under complement-positiveconditions (bar 10) (FIG. 14, Panel A). These effects were abolished inthe presence of complement when upon treatment with the SMR mutantpeptides (bars 9,11) (FIG. 14, Panel A).

In MDA-MB-231 cells treated with PEG-SMRwt-Clu, cell death increasedfrom 3.6% under complement-negative conditions (bar 4) to 76% undercomplement-positive conditions (bar 8)(FIG. 14, Panel B); likewise, inMDA-MB-231 cells treated with SMRwt-CPP, cell death increased from 3.6%under complement-negative conditions (bar 6) to 9% undercomplement-positive conditions (bar 10) (FIG. 14, Panel B). Theseeffects were abolished in the presence of complement when upon treatmentwith the SMR mutant peptides (bars 9,11) (FIG. 14, Panel B).

In K562 cells treated with PEG-SMRwt-Clu, cell death increased from 4.3%under complement-negative conditions (bar 4) to 55% undercomplement-positive conditions (bar 8)(FIG. 14, Panel C); likewise, inK562 cells treated with SMRwt-CPP, cell death increased from 4.3% undercomplement-negative conditions (bar 6) to 23% under complement-positiveconditions (bar 10) (FIG. 14, Panel C). These effects were abolished inthe presence of complement when upon treatment with the SMR mutantpeptides (bars 9,11) (FIG. 14, Panel C).

Taken together, these data show that SMR-induced mortalin sequestrationand functional disruption is linked to complement-mediated sensitivityand cell death. Specifically, blocking mortalin function renders orenhances tumor cell sensitivity to complement-mediated cytotoxicity.

Example 13: Mortalin Small Interfering RNA (siRNA) Reduces ExosomeSecretion and Induces Complement-Mediated Cytotoxicity

siRNA knockdown of mortalin expression was employed to further evaluatethe role of mortalin in reducing exosome secretion and protecting tumorcells from complement-mediated cytotoxicity. Briefly, MCF-7, MDA-MB-231and K562 cells were cultured in exosome-free media at 37° C. for 24 hr.The cells were then transfected with a vector expressing double-strandedmortalin siRNAs (Shelton et al., J Virol (2012) 86(1): p. 406-19; Huanget al., Oncotarget (2017) 8(7): p. 11302-11315). Negative control cellswere transfected with a vector expressing a double-stranded siRNApredicted not to target any known vertebrate gene.

Detection of mortalin protein in cells was measured by SDS-PAGE/Westernblot analysis using anti-mortalin and anti-α-tubulin (internal control)antibodies (FIG. 15, Panel A). Quantitative results from Western blotswere obtained by densitometry analysis of relative band intensities(FIG. 15, Panel B). Compared to the levels of mortalin expression incells transfected with the negative control, transfection of themortalin-targeted siRNA resulted in mortalin expression levels of:21.86% in MCF-7 (FIG. 15, Panel B, left sub-panel), 6.28% in MDA-MB-231(FIG. 15, Panel B, middle sub-panel) and 35.3% in K562 (FIG. 15, PanelC, right sub-panel).

In addition, exosomes were isolated from the cell culture supernatants,and analyzed for concentration, and size distribution via NanoSight LM10Nanoparticle Tracking Analysis (NTA). With NTA, particles areautomatically tracked and sized, based on Brownian motion and theassociated diffusion coefficient. Before analysis of the samples by NTA,it was determined that salt aggregates from PBS did not contribute tobackground and that equipment was free of contaminant particles.

The results of this analysis are shown in FIG. 15, Panel C. In MCF-7cells treated with the siRNA-Neg control, the cell culture supernatantwas found to contain 3.91×10⁹ particles/mL, while the mortalin siRNAtreated cells were found to contain 5.79×10′ particles/mL(p<2.02E-07)(FIG. 15, Panel C, left sub-panel). In MDA-MB-231 cellstreated with the siRNA-Neg control, the cell culture supernatant wasfound to contain 5.71×10⁹ particles/ml, while the mortalin siRNA treatedcells were found to contain 3.46×10⁹ particles/mL (p<1.82E-06)(FIG. 15,Panel C, middle sub-panel). In K562 cells treated with the siRNA-Negcontrol, the cell culture supernatant was found to contain 3.44×10⁹particles/mL, while the mortalin siRNA treated cells were found tocontain 2.58×10′ particles/mL (p<2.49E-06)(FIG. 15, Panel C, rightsub-panel). In all three cultures, NTA estimated the size of theexosomes to be in the range of 30 to 47 nm (data not shown).

To further validate the ability of mortalin neutralization to render orenhance tumor cell sensitivity to complement-mediated cytotoxicity,MCF-7, MDA-MB-231 and K562 cells were transfected with vectorsexpressing mortalin siRNAs or negative control siRNAs, incubated for 72hours at 37° C., treated with anti-CXCR4 antibody for 30 minutes at 4°C. followed by incubation with either NHS or NIS for 60 minutes at 37°C. The data from these experiments indicated that silencing of mortalinexpression resulted in mortalin-mediated protection of cells fromcomplement-dependent cytotoxicity at 38.02% relative to the negativecontrol in MCF-7 cells (FIG. 15, Panel D, left sub-panel), at 40.55%relative to the negative control in MDA-MB-231 cells (FIG. 15, Panel D,middle sub-panel) and at 30.54% relative to the negative control in K562cells (FIG. 15, Panel D, right subpanel).

Example 14: PEG-SMRwt-CLU Peptide Affect Migration of MCF-7 andMDA-MB-231 Breast Cancer Cells Migration, but not Non-TumorigenicMCF-10A

Cell migration or invasion is the fatal step in cancer progression andmetastasis, and accounts for 90% of all human cancer mortalities. Inaddition, cell migration is central to a variety of different pathologicand physiologic processes across many disciplines of biology includingwound healing, inflammation, cell growth and differentiation. Cellinvasion refers to 3-dimensional migration of cells as they penetrate anextracellular matrix (ECM) and is a process typically associated withcancer cell metastasis. To further explore this process, it was ofinterest to determine whether SMRwt peptides inhibit breast cancer cellmigration and invasion. Consequently, a migration assay was performed toexamine whether SMRwt peptides blocked tumor cells migration.

Specifically, MDA-MB-231 and MCF-7 breast cancer cells were treated with1120 nM SMR peptide in MCF-7 cells, 280 nM SMR peptide in MDA-MB-231cells or 3 μM Latrunculin A (positive control; known migratoryinhibitor) for 24 hours and cell migration assays were performedaccording to manufacturer's instructions (Calbiochem manual) using afluorescence plate reader set at an excitation wavelength of 485 nm andan emission wavelength of 515 nm.

In MCF-7 cells, the SMRwt-CPP peptide, PEG-SMRwt-CLU peptide, andlatrunculin A (the positive inhibitor control) reduced migration by71.87%, 75.74%, and 53.05%, respectively (FIG. 16A). In MDA-MB-231cells, the SMRwt-CPP peptide, PEG-SMRwt-CLU peptide, and latrunculin Areduced migration by 46.39%, 52.37%, and 50.39%, respectively (FIG.16B). Further, the effects were specific for wild-type SMR peptides, asthere was no significant inhibition of migration observed followingtreatment with mutant SMR peptides. In contrast, in MCF-10A, the normalbreast cell line, migration was not significantly affected by any of thepeptides or the inhibitor (FIG. 16C). Taken together, these data showthat the SMRwt-CPP and PEG-SMRwt-CLU peptides significantly reducedbreast cancer cell migration and invasion, while mutated SMR peptideshad no statistically significant effect.

Example 15. Summary of Observations and Conclusions from Examples 8-14

A series of peptides derived from the Secretion Modification Region(SMR) of HIV-1 Nef protein were modified by addition of either acell-penetrating peptide (CPP), a positively charged arginine-richpeptide derived from HIV-1 regulatory protein Tat, or a Clusterin (CLU)peptide, a molecular chaperone involved in protein secretion. Both CPPand CLU peptides were added at the C-terminus of the Nef SMR peptide.The CLU peptides were also modified with polyethylene glycol (PEG) toenhance solubility. After treatment of cells with the peptides, MTT cellviability and migration assays were used to confirm the inhibitoryeffect of these modified SMRwt peptides on the proliferation ofMDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flowcytometry was used to determine complement mediated cell apoptosis anddeath. Western blot analysis was used to track peptide-mediated changesin expression of mortalin protein in both treated cells and exosomesreleased from the treated cells. NanoSight analyses andacetylcholinesterase (AChE) assays were employed for measuring exosomeparticle sizes and exosome concentrations in the media.

To investigate a functional role for mortalin in breast cancer andleukemia cells, mortalin knockdown experiments were conducted in MCF-7and MDA-MB-231 breast cancer cells and leukemia cells and a series ofassays were performed to examine the effects of mortalin expressionand/or sequestration on cancer cell proliferation and metastasis. SMRwtpeptides interacted with mortalin to significantly reduce cellproliferation and to inhibit cancer cell growth and cellmigration/invasion. The oncogenic functions possessed by mortalin appearto be closely related in both breast cancer and leukemia cells. Inparticular, SMRwt peptides were found to interfere with the ability ofmortalin to promote and protect cancer progression. First, the modifiedSMRwt peptides reduced the expression of the mesenchymal marker vimentin(VIM). Second, exposure to SMRwt peptides inhibited migration of breastcancer cells as measured by the migration assay. Third, the SMRwtpeptides blocked the ability of cancer cells to release exosomes, whichin turn blocked exosome-mediated release of complement, therebyre-establishing complement mediated cell death in those peptide-treatedcells.

The data herein further show that: (i) SMRwt peptides not onlyeffectively inhibited the growth of MDA-MB-231 and MCF-7 breast cancercells but also unexpectedly inhibited the growth of a second, unrelatedtumor line, K562 leukemia cells; (ii) Mortalin-SMR peptide interactionsin cancer cells showed increased apoptosis, consistent with SMRwtpeptide interfering with resistance of cancer cells tocomplement-dependent cytotoxicity; (iii) siRNA-mediated knockdown ofmortalin reduced membrane attack complex (MAC) elimination and enhancedcell sensitivity to MAC-induced cell death; (iv) SMRwt peptides and themortalin inhibitors, MKT-077, Omeprazole and 5-(N,N-Dimethyl)amiloride(DMA) blocked exosome secretion in MCF-7 and MDA-MB-231 breast cancerand K562 leukemia cells; and (v) PEG-SMRwt peptides inhibited MCF-7 andMDA-MB-231 breast cancer cell migration by blocking exosome release.

In conclusion, the data suggests mortalin promotes cell proliferation,invasion, and resistance to complement mediated cell death via inductionof epithelial mesenchymal transition (EMT), for example, in leukemiacells. SMRwt peptides antagonize the functions of mortalin, blockingtumor exosome release and exosome-mediated release of complement, andantagonizing tumor cell migration and invasion associated with EMT.Treatment of cancer cells with these peptides alone or in combinationwith other active agents can reduce breast cancer and/or leukemia cellinvasion and metastasis and faciliate standard treatment of these latestage tumor cells. The findings presented herein have important clinicalimplications and support further investigation into the therapeuticvalue of SMR peptides for treatment of cancer.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present disclosure, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentembodiment, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence thatis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A multipartite peptide, comprising the amino acidsequence of NXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 37).
 2. Themultipartite peptide of claim 1, further comprising a signal peptidedomain for secretion of the multipartite peptide from cells expressingthe multipartite peptide.
 3. A polynucleotide encodingNXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 37).
 4. An expression vector,comprising: a polynucleotide encoding NXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQID NO: 37); and a regulatory sequence operably linked to thepolynucleotide.
 5. A pharmaceutical composition comprising amultipartite peptide comprising the amino acid sequence ofNXNVGFPVAAVGFPVHPLSKHPYWSQP (SEQ ID NO: 37), wherein the peptide isassociated with a liposome as a carrier.